Plant artificial chromosome (PLAC) compositions and methods

ABSTRACT

The present invention provides for the identification and cloning of functional plant centromeres in  Arabidopsis . This will permit construction of stably inherited plant artificial chromosomes (PLACs) which can serve as vectors for the construction of transgenic plant and animal cells. In addition, information on the structure and function of these regions will prove valuable in isolating additional centromeric and centromere related genetic elements and polypeptides from other species.

This application is a continuation of U.S. patent application Ser. No.10/161,849, filed Aug. 6, 2002, which is a continuation of U.S. patentapplication Ser. No. 09/553,231, filed Apr. 19, 2000, now issued as U.S.Pat. No. 6,900,012, which is a continuation of U.S. patent applicationSer. No. 09/090,051, filed Jun. 3, 1998, now U.S. Pat. No. 6,156,953,which claims the priority of U.S. Provisional Patent Application Ser.No. 60/048,451, filed Jun. 3, 1997; and U.S. Provisional PatentApplication Ser. No. 60/073,741, filed Feb. 5, 1998, both of thedisclosures of which are specifically incorporated herein by referencein their entirety.

The government owns rights in the present invention pursuant to U.S.Department of Agriculture Grant No. 96-35304-3491 and Grant No.DE-FC05-920R22072 from the Consortium for Plant Biotechnology.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the field of molecularbiology. More particularly, it concerns the construction and use ofplant artificial chromosomes.

II. Description of Related Art

Two general approaches are used for introduction of new geneticinformation (“transformation”) into cells. One approach is to introducethe new genetic information as part of another DNA molecule, referred toas a “vector,” which can be maintained as an independent unit (anepisome) apart from the chromosomal DNA molecule(s). Episomal vectorscontain all the necessary DNA sequence elements required for DNAreplication and maintenance of the vector within the cell. Many episomalvectors are available for use in bacterial cells (for example, seeManiatis et al., 1982). However, only a few episomal vectors thatfunction in higher eukaryotic cells have been developed. The availablehigher eukaryotic episomal vectors are based on naturally occurringviruses and most function only in mammalian cells (Willard, 1997). Inhigher plant systems the only known double-stranded DNA viruses thatreplicate through a double-stranded intermediate upon which an episomalvector could be based is the gemini virus, although the gemini virus islimited to an approximately 800 bp insert. Although an episomal plantvector based on the Cauliflower Mosaic Virus has been developed, itscapacity to carry new genetic information also is limited (Brisson etal., 1984).

The other general method of genetic transformation involves integrationof introduced DNA sequences into the recipient cell's chromosomes,permitting the new information to be replicated and partitioned to thecell's progeny as a part of the natural chromosomes. The most commonform of integrative transformation is called “transfection” and isfrequently used in mammalian cell culture, systems. Transfectioninvolves introduction of relatively large quantities of deproteinizedDNA into cells. The introduced DNA usually is broken and joined togetherin various combinations before it is integrated at random sites into thecell's chromosome (see, for example Wigler et al., 1977). Commonproblems with this procedure are the rearrangement of introduced DNAsequences and unpredictable levels of expression due to the location ofthe transgene in the genome or so called “position effect variation”(Shingo et al., 1986). Further, unlike episomal DNA, integrated DNAcannot normally be precisely removed. A more refined form of integrativetransformation can be achieved by exploiting naturally occurring virusesthat integrate into the host's chromosomes as part of their life cycle,such as retroviruses (see Cepko et al., 1984). In mouse, homologousintegration has recently become common, although it is significantlymore difficult to use in plants (Lam et al. 1996).

The most common genetic transformation method used in higher plants isbased on the transfer of bacterial DNA into plant chromosomes thatoccurs during infection by the phytopathogenic soil bacteriumAgrobacterium (see Nester et al., 1984). By substituting genes ofinterest for the naturally transferred bacterial sequences (calledT-DNA), investigators have been able to introduce new DNA into plantcells. However, even this more “refined” integrative transformationsystem is limited in three major ways. First, DNA sequences introducedinto plant cells using the Agrobacterium T-DNA system are frequentlyrearranged (see Jones et al., 1987). Second, the expression of theintroduced DNA sequences varies between individual transformants (seeJones et al., 1985). This variability is presumably caused by rearrangedsequences and the influence of surrounding sequences in the plantchromosome (i.e., position effects), as well as methylation of thetransgene. A third drawback of the Agrobacterium T-DNA system is thereliance on a “gene addition” mechanism: the new genetic information isadded to the genome (i.e., all the genetic information a cell possesses)but does not replace information already present in the genome.

One attractive alternative to commonly used methods of transformation isthe use of an artificial chromosome. Artificial chromosomes are man-madelinear or circular DNA molecules constructed from essential cis-actingDNA sequence elements that are responsible for the proper replicationand partitioning of natural chromosomes (see Murray et al., 1983). Theessential elements are: (1) Autonomous Replication Sequences (ARS)(these have properties of replication origins, which are the sites forinitiation of DNA replication), (2) Centromeres (site of kinetochoreassembly and responsible for proper distribution of replicatedchromosomes at mitosis or meiosis), and (3) Telomeres (specializedstructures at the ends of linear chromosomes that function to stabilizethe ends and facilitate the complete replication of the extreme terminiof the DNA molecule).

At present, the essential chromosomal elements for construction ofartificial chromosomes have been precisely characterized only from lowereukaryotic species. ARSs have been isolated from unicellular fingi,including Saccharomyces cerevisiae(brewer's yeast) andSchizosaccharomyces pombe (see Stinchcomb et al., 1979 and Hsiao et al.,1979). ARSs behave like replication origins allowing DNA molecules thatcontain the ARS to be replicated as an episome after introduction intothe cell nuclei of these fungi. Plasmids containing these sequencesreplicate, but in the absence of a centromere they are partitionedrandomly into daughter cells.

Artificial chromosomes have been constructed in yeast using the threecloned essential chromosomal elements. Murray et al., 1983, disclose acloning system based on the in vitro construction of linear DNAmolecules that can be transformed into yeast, where they are maintainedas artificial chromosomes. These yeast artificial chromosomes (YACs)contain cloned genes, origins of replication, centromeres and telomeresand are segregated in daughter cells with high affinity when the YAC isat least 100 kB in length. Smaller CEN containing vectors may be stablysegregated, however, when in circular form.

None of the essential components identified in unicellular organisms,however, function in higher eukaryotic systems. For example, a yeast CENsequence will not confer stable inheritance upon vectors transformedinto higher eukaryotes. While such DNA fragments can be readily beintroduced, they do not stably exist as episomes in the host cell. Thishas seriously hampered efforts to produce artificial chromosomes inhigher organisms.

In one case, a plant artificial chromosome was discussed (Richards etal., U.S. Pat. No. 5,270,201). However, this vector was based on planttelomeres, as a functional plant centromere was not disclosed. Whiletelomeres are important in maintaining the stability of chromosomaltermini, they do not encode the information needed to ensure stableinheritance of an artificial chromosome. It is well documented thatcentromere function is crucial for stable chromosomal inheritance inalmost all eukaryotic organisms (reviewed in Nicklas 1988). For example,broken chromosomes that lack a centromere (acentric chromosomes) arerapidly lost from cell lines, while fragments that have a centromere arefaithfully segregated. The centromere accomplishes this by attaching,via centromere binding proteins, to the spindle fibers during mitosisand meiosis, thus ensuring proper gene segregation during celldivisions.

In contrast to the detailed studies done in S. cerevisiae and S. pombe,little is known about the molecular structure of functional centromericDNA of higher eukaryotes. Ultrastructural studies indicate that highereukaryotic kinetochores, which are specialized complexes of proteinsthat form on the chromosome during late prophase, are large structures(mammalian kinetochore plates are approximately 0.3 μm in diameter)which possess multiple microtubule attachment sites (reviewed in Rieder,1982). It is therefore possible that the centromeric DNA regions ofthese organisms will be corresponding large, although the minimal amountof DNA necessary for centromere function may be much smaller.

While the above studies have been useful in elucidating the structureand function of centromeres, they have failed to provide a cloned,functional centromere from a higher eukaryotic organism. The extensiveliterature indicating both the necessity of centromeres for stableinheritance of chromosomes, and the non-functionality of yeastcentromeres in higher organisms, demonstrate that cloning of afunctional centromere from a higher eukaryote is a necessary first stepin the production of artificial chromosomes suitable for use in higherplants and animals. The production of artificial chromosomes withcentromeres which function in higher eukaryotes would overcome many ofthe problems associated with the prior art and represent a significantbreakthrough in biotechnology research.

SUMMARY OF THE INVENTION

The current invention overcomes deficiencies in the prior art byproviding methods for obtaining a functional plant centromere and usestherefor. More particularly, the present invention provides for theproduction of a stably inherited plant artificial chromosome.

In a first embodiment, there is provided a method for the identificationplant centromeres. Briefly, tetrad analysis measures the recombinationfrequency between genetic makers and a centromere by analyzing all fourproducts of individual meiosis. A particular advantage arises from thequartet (qrt 1) mutation in Arabidopsis, which causes the four productsof pollen mother cell meiosis in Arabidopsis to remain attached. Whenused to pollinate a flower, one tetrad can result in the formation offour seeds, and the plants from these seeds can be analyzed genetically.With unordered tetrads, however, such as those produced by Arabidopsis,genetic mapping using tetrad analysis requires that two markers bescored simultaneously.

In another embodiment, the present invention provides new plantartificial chromosomes (PLACs) and DNA fragments for the creationthereof. In a preferred embodiment the PLAC will have functionalsequences which include telomeres, a plant and/or other autonomousreplicating sequence, a centromere, and selectable markers which confera growth advantage under particular conditions to plant cells carryingthe marker, thereby allowing identification of plants, plants cells orcells from any other organism of interest containing the PLAC. Aselectable marker may in particular embodiments of the inventionfunction in bacterial cells. The PLAC also may contain “negative”selectable markers which confer susceptibility to an antibiotic,herbicide or other agent, thereby allowing for selection against plants,plant cells or cells of any other organism of interest containing aPLAC. The PLAC also may include genes which control the copy number ofthe PLAC within a cell. One or more structural genes also may beincluded in the PLAC. Specifically contemplated as being useful will beas many structural genes as may be inserted into the PLAC while stillmaintaining a functional vector. This may include one, two, three, four,five, six, seven, eight, nine or more structural genes.

In another embodiment, the invention provides methods for expressingforeign genes in plants, plant cells or cells of any other organism ofinterest. The foreign genes may be from any organism, including plants,animals and bacteria. It is further contemplated that PLACs could beused to simultaneously transfer multiple foreign genes to a plantcomprising entire biochemical or regulatory pathways. In yet anotherembodiment of the invention, it is contemplated that the PLACs can beused as DNA cloning vectors. Such a vector could be used in plant andanimal sequencing projects. The current invention may be of particularuse in the cloning of sequences which are “unclonable” in yeast andbacteria, but which may be easier to clone in a plant based system.

In still yet another embodiment of the invention, it is contemplatedthat the PLACs disclosed herein may be used clone functional segments ofDNA such as origins of DNA replication, telomeres, telomere associatedgenes, nuclear matrix attachment regions (MARs), scaffold attachmentregions (SARs), boundary elements, enhancers, silencers, promoters,recombinational hot-spots and centromeres. This embodiment may becarried out by cloning DNA into a defective PLAC which is deficient forone or more type of functional elements. Sequences which complementedsuch deficient elements would cause the PLAC to be stably inherited. Aselectable marker on the PLAC could then be used to select for viablePLAC containing cells which contain cloned functional elements of thetype that were non-functional in the defective PLAC.

In still yet another embodiment of the invention, the sequencesdisclosed herein may be used for the isolation of centromeric sequencesfrom other plant species including agriculturally important species suchas Brassica species (Broccoli, Cauliflower, Mustard, etc.), and monocotssuch as wheat and corn. Methods for isolating centromeric sequencesusing the sequences disclosed herein are well known in the art and arebased on shared homology of the centromeric sequences.

In still yet another embodiment of the invention, the artificialchromosome vectors described herein may be used to perform efficientgene replacement studies. At present, gene replacement has been detectedon only a few occasions in plant systems and has only been detected atlow frequency in mammalian tissue culture systems (see Thomas et al.,1986; Smithies et al., 1985). The reason for this is the high frequencyof illegitimate nonhomologous recombination events relative to thefrequency of homologous recombination events (the latter are responsiblefor gene replacement). Artificial chromosomes may participate inhomologous recombination preferentially. Since the artificialchromosomes remain intact upon delivery, no recombinogenic broken endswill be generated to serve as substrates for the extremely efficientillegitimate recombination machinery. Thus, the artificial chromosomevectors disclosed by the present invention will be stably maintained inthe nucleus through meiosis and available to participate inhomology-dependent meiotic recombination. In addition, because inprinciple, artificial chromosomes of any length could be constructedusing the teaching of the present invention, the vectors could be usedto introduce extremely long stretches of DNA from the same or any otherorganism into cells. Specifically contemplated inserts include thosefrom about several base pairs to one hundred megabase pairs, includingabout 1 kb, 25 kB, 50 kB, 100 kB, 125 kB, 150 kB, 200 kB, 300 kB, 400kB, 500 kB, 600 kB, 700 kB, 800 kB, 900 kB, 1 MB, 1.25 Mb, 1.5 Mb, 2 Mb,3 Mb, 5 Mb, 10 Mb, 25 Mb, 50 Mb and 100 Mb.

In still yet another embodiment, the present invention relates to theconstruction of artificial chromosome vectors for the genetictransformation of plant cells, processes for their preparation, uses ofthe vectors, and organisms transformed by them. Standard reference workssetting forth the general principles of recombinant DNA technologyinclude Lewin, 1985. Other works describe methods and products ofgenetic engineering; see, e.g., Maniatis et al., 1982; Watson et al.,1983; Setlow et al., 1979; and Dillon et al., 1985.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Centromere mapping with unordered tetrads: A cross of twoparents (AABB×aabb), in which “A” is on the centromere of onechromosome, and “B” is linked to the centromere of a second chromosome.At meiosis, the A and B chromosomes assort independently, resulting inequivalent numbers of parental ditype (PD) and nonparental ditype (NPD)tetrads (recombinant progeny are shown in gray). Tetratype tetrads (TT)result only from a crossover between “B” and the centromere.

FIG. 2: Low resolution map location of Arabidopsis centromeres. Trisomicmapping was used to determine the map position of centromeres on four ofthe five Arabidopsis chromosomes (Koornneef 1983; Sears et al, 1970).For chromosome 4, useful trisomic strains were not obtained. With themethods of Koornneef and Sears et al, 1983. (which rely onlow-resolution deletion mapping) the centromere on chromosome 1 wasfound to lie between the two visible markers, tt1 and ch1, that areseparated by 5 cM. Centromere positions on the other chromosomes aremapped to a lower resolution.

FIG. 3A-E: Map location of Arabidopsis thaliana centromeres onchromosomes 1-5. Centromeres were mapped by tetrad analysis as describedbelow. Genetic markers used to map the centromeres are displayed abovethe chromosomes and are designated by their cM values (all cM valueswere derived from the Lister and Dean recombinant inbred map at the website: http://genome-www3.stanford.edu/atdb_welcome.html).

FIG. 4: Seed stock used for tetrad analysis in Arabidopsis thaliana. Theindividual strains are identified by the strain number. The tetradmember number indicates the tetrad source (i.e. T1 indicates seeds fromtetrad number 1, and the numbers −1, −2, −3, or −4 indicate individualmembers of the tetrad). The strains listed have been deposited with theArabidopsis Biological Resources Center (ABRC) at Ohio State Universityunder the name of Daphne Preuss, or where seed set has not yet occurred,will be deposited when collected (indicated in column 3).

FIG. 5: Marker information for centromere mapping. DNA polymorphismsused to localize the centromeres are indicated by chromosome (Column 1).The name of each marker is shown in Column 2, and the marker type inColumn 3. CAPS (Co-dominant Amplified Polymorphic Sites) are markersthat can be amplified with PCR and detected by digesting with theappropriate restriction enzyme (also indicated in Column 3). SSLPs(Simple Sequence Length Polymorphisms) detect polymorphisms byamplifying different length PCR products. Column 4 notes if the markeris available on public web sites(http://genome-www.stanford.edu/Arabidopsis). For those markers that arenot available on public web sites the sequences of the forward andreverse primers used to amplify the marker are listed in columns 5 and6, respectively, and given in SEQ ID NO:1 through SEQ ID NO:44.

FIG. 6: Scoring PCR-based markers for tetrad analysis. The genotype ofthe progeny from one pollen tetrad (T2) was determined for two geneticmarkers (SO392 and nga76). Analysis of the four progeny plants (T2-1through T2-4) using PCR and gel electrophoresis allows the genotype ofthe plant to be determined, and the genotype of the pollen parent to beinferred.

FIGS. 7A-7H: Exemplary PLAC vectors: The vectors shown in FIG. 7A, FIG.7B, FIG. 7E, and FIG. 7F have an E. coli origin of replication which canbe high copy number, low copy number or single copy. In FIGS. 7A-7H, thevectors include a multiple cloning site which can contain recognitionsequences for conventional restriction endonucleases with 4-8 bpspecificity as well as recognition sequences for very rare cuttingenzymes such as, for example, I-Ppo I, I-Ceu I, PI-Tli I, PI-Psp I, NotI, and PI See I. In FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG.7G, and FIG. 7H the centromere is flanked by Lox sites which can act astargets for the site specific recombinase Cre. FIG. 7A: An E. coli plantcircular shuttle vector with a plant ARS. FIG. 7B: A plant circularshuttle vector without a plant ARS. The vector relies on a plant originof replication function found in other plant DNA sequences such asselectable markers. FIG. 7C: A yeast-plant circular shuttle vector witha plant ARS. The yeast ARS is included twice, once on either side of themultiple cloning site to ensure that large inserts are stable. FIG. 7D:A yeast-plant circular shuttle vector without a plant ARS. The vectorrelies on a plant origin of replication function found in other plantDNA sequences such as selectable markers. The yeast ARS is includedtwice, once on either side of the multiple cloning site to ensure thatlarge inserts are stable. FIG. 7E: An E. coli—Agrobacterium—Plantcircular shuttle vector with a plant ARS. Vir functions for T-DNAtransfer would be provided in trans by using the appropriateAgrobacterium strain. FIG. 7F: An E. coli—Agrobacterium—plant circularshuttle vector without a plant ARS. The vector relies on the plantorigin of replication function found in other plant DNA sequences suchas selectable markers. Vir functions for T-DNA transfer would beprovided in trans by using the appropriate Agrobacterium strain. FIG.7G: A linear plant vector with a plant ARS. The linear vector could beassembled in vitro and then transferred into the plant by, for example,mechanical means such as microprojectile bombardment, electroporation,or PEG-mediated transformation. FIG. 7H: A linear plant vector without aplant ARS. The linear vector could be assembled in vitro and thentransferred into the plant by, for example, mechanical means suchmicroprojectile bombardment, electroporation, and PEG-mediatedtransformation.

DETAILED DESCRIPTION OF THE INVENTION

The prior art has failed to provide a centromere which is functional inplants. This failure is exemplified by the general lack of detailedinformation in the art regarding the centromeres of multicellularorganisms in general. To date, the most extensive and reliablecharacterization of centromere sequences has come from studies of lowereukaryotes such as S. cerevisiae and S. pombe, where the ability toanalyze centromere functions has provided a clear picture of theessential DNA sequences. The S. cerevisiae centromere consists of threeessential regions, CDEI, CDEII, and CDEIII, totaling only 125 bp, orapproximately 0.006 to 0.06% of each yeast chromosome (Carbon et al.,1990; Bloom 1993). S. pombe centromeres are between 40 and 100 kB inlength and consist of repetitive elements that comprise 1 to 3% of eachchromosome (Baum et al., 1994). Subsequent studies, using tetradanalysis to follow the segregation of artificial chromosomes,demonstrated that less than ⅕ of the naturally occurring S. pombecentromere is sufficient for centromere function (Baum et al., 1994).

In contrast, the centromeres of mammals and other higher eukaryotes arepoorly defined. Although DNA fragments that hybridize to centromericregions in higher eukaryotes have been identified, little is knownregarding the functionality of these sequences (see Tyler-Smith et al.,1993). In many cases centromere repeats correlate with centromerelocation, with probes to the repeats mapping both cytologically andgenetically to centromere regions. Many of these sequences aretandemly-repeated satellite elements and dispersed repeated sequences inarrays ranging from 300 kB to 5000 kB in length (Willard 1990). To date,only one of these repeats, a 171 bp element known as the alphoidsatellite, has been shown by in situ hybridization to be present at eachhuman centromere (Tyler-Smith et al., 1993). Whether repeats themselvesrepresent functional centromeres remains controversial, as other genomicDNA is required to confer inheritance upon a region of DNA (Willard,1997). Alternatively, the positions of some higher eukaryoticcentromeres have been estimated by analyzing the segregation ofchromosome fragments. This approach is imprecise, however, because alimited set of fragments can be obtained, and because normal centromerefunction is influenced by surrounding chromosomal sequences (forexample, see Koornneef, 1983; FIG. 2).

A more precise method for mapping centromeres that can be used in intactchromosomes is tetrad analysis (Mortimer et al., 1981), which provides afunctional definition of a centromere in its native chromosomal context.At present, the only centromeres that have been mapped in this mannerare from unicellular eukaryotes, including the yeasts Saccharomycescerevisiae, Schizosaccharomyces pombe, and Kluyveromyces lactis (Carbonet al, 1990; Hegemann et al., 1993). In these systems, accurate mappingof the centromeres made it possible to clone centromeric DNA, using achromosome walking strategy (Clarke et al., 1980). Subsequently,artificial chromosome assays were used to define more precisely thecentromere sequences (Hegemann et al., 1993; Baum et al., 1994).

Attempts to develop a reliable centromeric assay in mammals have yieldedambiguous results. For example, Hadlaczky et al., (1991) identified a 14kB human fragment that can, at low frequency, result in de novocentromere formation in a mouse cell line. In situ hybridizationstudies, however, have shown that this fragment is absent from naturallyoccurring centromeres, calling into question the reliability of thisapproach for testing centromere function (Tyler-Smith et al., 1993).Similarly, transfection of alphoid satellites into cell lines results inthe formation of new chromosomes, yet these chromosomes also containhost sequences that could contribute centromere activity (Haaf et al.,1992; Willard, 1997). Further, the novel chromosomes can have alphoidDNA spread throughout their length yet have only a single centromericconstriction, indicating that a block of alphoid DNA alone may beinsufficient for centromere function (Tyler-Smith et al., 1993).

Although plant centromeres can be visualized easily in condensedchromosomes, they have not been characterized as extensively ascentromeres from yeast or mammals. Genetic characterization has reliedon segregation analysis of chromosome fragments, and in particular onanalysis of trisomic strains that carry a genetically marked,telocentric fragment (for example, see Koornneef 1983; FIG. 2). Inaddition, repetitive elements have been identified that are eithergenetically (Richards et al., 1991) or physically (Alfenito et al.,1993; Maluszynska et al., 1991) linked to a centromere. In no case,however, has the functional significance of these sequences been tested.

Cytology in Arabidopsis thaliana has served to correlate centromerestructure with repeat sequences. A fluorescent dye, DAPI, allowsvisualization of centromeric chromatin domains in metaphase chromosomes.A fluorescence in situ hybridization (FISH) probe based on 180 bp pAL1repeat sequences colocalized with the DAPI signature near thecentromeres of all five Arabidopsis chromosomes (Maluszynska et al.,1991; Martinez-Zapater et al., 1986), however this repeat probe alsohybridizes within noncentromeric regions of the chromosomes. Although afunctional role for pAL1 has been proposed, more recent studies havefailed to detect this sequence near the centromeres in species closelyrelated to Arabidopsis thaliana (Maluszynska et al., 1993). Theseresults are particularly troubling because one of the species tested, A.pumila, is thought be an amphidiploid, derived from a cross between A.thaliana and another close relative (Maluszynska et al., 1991, Price etal., 1995). Another repetitive sequence, pAtT12, has been geneticallymapped to within 5 cM of the centromere on chromosome 1 and to thecentral region of chromosome 5 (Richards et al., 1991), although itspresence on other chromosomes has not been established. Like pAL1, arole for pAtT12 in centromere function remains to be demonstrated.

Due to the fact that kinetochores constitute a necessary link betweencentromeric DNA and the spindle apparatus, the proteins that areassociated with these structures recently have been the focus of intenseinvestigation (Bloom 1993; Earnshaw 1991). Human autoantibodies thatbind specifically in the vicinity of the centromere have facilitated thecloning of centromere-associated proteins (CENPs, Rattner 1991), and atleast one of these proteins belongs to the kinesin superfamily ofmicrotubule-based motors (Yen 1991). Yeast centromere-binding proteinsalso have been identified, both through genetic and biochemical studies(Bloom 1993; Lechner et al., 1991). Although the basic features ofmitosis and meiosis (including spindle attachment, chromosome pairing,and chromosome separation) are conserved among all eukaryotes, acomparison of the known centromere-binding proteins from yeast andhumans reveals little similarity (Bloom, 1993; Earnshaw and Cooke,1989).

The centromeres of Arabidopsis thaliana have been mapped using trisomicstrains, where the segregation of chromosome fragments (Koornneef 1983)or whole chromosomes (Sears et al., 1970) was used to localize four ofthe centromeres to within 5, 12, 17 and 38 cM, respectively (FIG. 2).These positions have not been refined by more recent studies because themethod is limited, not only by the difficulty in obtaining viabletrisomic strains, but also by the inaccuracy of comparing recombinationfrequencies in chromosome fragments to those in intact chromosomes(Koornneef 1983). These factors introduce significant error into thecalculated position of the centromere, and in Arabidopsis, where 1 cMcorresponds roughly to 200 kB (Kornneef 1987; Hwang et al., 1991), thismethod did not map any of the centromeres with sufficient precision tomake chromosome waking strategies practical.

I. TETRAD ANALYSIS

With tetrad analysis, the recombination frequency between geneticmarkers and a centromere can be measured directly (FIG. 1). This methodrequires analysis of all four products of individual meiosis, and it hasnot been applied previously to multicellular eukaryotes because theirmeiotic products typically are dissociated. Identification of thequartet mutation makes tetrad analysis possible for the first time in amulticellular genetic model system (Preuss et al., 1994). The quartet(qrt 1) mutation causes the four products of pollen mother cell meiosisin Arabidopsis to remain attached. When used to pollinate a flower, onetetrad can result in the formation of four seeds, and the plants fromthese seeds can be analyzed genetically.

With unordered tetrads, such as those produced by S. cerevisiae orArabidopsis, genetic mapping using tetrad analysis requires that twomarkers be scored simultaneously (Whitehouse 1950). Tetrads fall intodifferent classes depending on whether the markers are in a parental(nonrecombinant) or nonparental (recombinant) configuration (FIG. 1). Atetrad with only nonrecombinant members is referred to as a parentalditype (PD); one with only recombinant members as a nonparental ditype(NPD); and a tetrad with two recombinant and two nonrecombinant membersas a tetratype (TT) (Perkins 1953). If two genetic loci are on differentchromosomes, and thus assort independently, the frequency of tetratype(crossover products) versus parental or nonparental assortment ditype(noncrossover products) depends on the frequency of crossover betweeneach of the two loci and their respective centromeres.

Tetratype tetrads arise only when a crossover has occurred between amarker in question and its centromere. Thus, to identify genes that areclosely linked to the centromere, markers are examined in a pair-wisefashion until the TT frequency approaches zero. The genetic distance (incentimorgans, cM) between the markers and their respective centromeresis defined by the function [(½)TT]/100 (Mortimer et al., 1981). Becausepositional information obtained by tetrad analysis is a representationof physical distance between two points, as one approaches thecentromere the chance of a recombination event declines.

Tetrad analysis has been used to genetically track centromeres in yeastsand other fungi in which products of a single meioses can be collected.The budding yeast Saccharomyces cerevisiae lacks mitotic condensationand thus cytogenetics (Hegemann et al., 1993), yet due to tetradanalysis, has served as the vehicle of discovery for centromerefunction. Meiosis is followed by the generation of four spores heldwithin an ascus and these can be directly assayed for gene segregation.

The recessive qrt1 mutation makes it possible to perform tetrad analysisin Arabidopsis by causing the four products of meiosis to remainattached (Preuss et al., 1994; and Smythe 1994; both incorporated hereinby reference). As previously shown, within each tetrad, genetic locisegregate in a 2:2 ratio (FIG. 6). Individual tetrads can be manipulatedonto flowers with a fine brush (at a rate of 20 tetrads per hour), andin 30% of such crosses, four viable seeds can be obtained (Preuss etal., 1994).

Mapping centromeres with high precision requires a dense genetic map,and although the current Arabidopsis map contains many visible markers,it would be laborious to cross each into the qrt1 background.Alternatively, hundreds of DNA polymorphisms can be introducedsimultaneously by crossing two different strains, both containing theqrt1 mutation. A dense RFLP map (Chang et al., 1988) and PCR-based maps(Konieczny et al., 1993; Bell et al., 1994) have been generated inArabidopsis from crosses of the Landsberg and Columbia strains(Arabidopsis map and genetic marker data is available from the internetat http://genome-www.stanford.edu/Arabidopsis andhttp://cbil.humgen.upenn.edu/atgc/sslp_info/sslp.html). These strainsdiffer by 1% at the DNA sequence level and have colinear genetic maps(Chang et al., 1988; Koornneef, 1987).

Centromere mapping with tetrad analysis requires simultaneous analysisof two markers, one of which must be centromere-linked (FIG. 1). Toidentify these centromere-linked markers, markers distributed across all5 chromosomes were scored and compared in a pairwise fashion.

Initially, genetic markers that can be scored by PCR analysis weretested (Konieczny et al., 1993; Bell et al., 1994). Such markers aresufficiently dense to map any locus to ±10 cM, and as additionalPCR-detectable polymorphisms are identified they are incorporated intothe analyses. As higher resolution of mapping becomes necessary,selected RFLPS, from the existing set of over 350 markers will bescored. In addition, as described in FIG. 5, new CAPS and SSLP markersuseful for mapping the centromere can be readily identified.

II. TETRAD SETS

To date, progeny plants from 388 isolated tetrad seed sets have beengerminated and leaf tissue collected and stored from each of the tetradprogeny plants. The leaf tissue from individual plants was used to makeDNA for PCR based marker analysis. The plants also were allowed toself-fertilize and the seed they produced was collected (a list of theseed stock of informative individuals used for tetrad analysis is givenin FIG. 4). From each of these individual seed sets, seedlings can begerminated and their tissues utilized for making genomic DNA. Tissuepooled from multiple seedlings is useful for making Southern genomic DNAblots for the analysis of restriction fragment length polymorphisms(RFLPs). Informative plants which have been used for tetrad analysis aregiven in FIG. 4.

III. MAPPING STRATEGY

The initial mapping of the centromeres was completed with selected PCR™based markers (such as CAPS and SSLPs) (FIG. 5). These markers wereselected from the large number of markers available for Arabidopsis andare distributed across the genetic map. Due to the fact that analysis ofRFLP markers is labor intensive, those markers that could be analyzedwith PCR™ based markers were surveyed first. Only a subset of theoriginal 388 tetrads have provided crossovers close enough to theircentromeres as to require the use of RFLP marker segregation analysis.For those tetrads in which existing markers do not identify the regionof crossover between centromere and marker, new markers may bedeveloped. This may be accomplished by screening cloned sequences inregions of interest for new potential markers, i.e., RFLPs, CAPS, SSLPs,and the like. The new markers may then be used to generate new data withthe relevant tetrads. In addition to the markers in centromeric regions,the segregation of two markers per chromosome that are both, far from,and flank the centromeric regions were assessed. Data from these markerswill often be tetratype and thus identify whether a Landsberg toColumbia crossover or a Columbia to Landsberg crossover is visible asthe centromere is approached.

Codominant cleaved amplified polymorphic sequences (CAPS) were amplifiedby PCR™. The PCR™ products were observed on agarose gels prior tocleavage by restriction enzymes. Once cleaved, the products were scoredfor marker segregation on agarose or acrylamide gels. Simple sequencelength polymorphisms (SSLPs) also were amplified by PCR™. The PCR™products were directly scored by polyacrylamide gel electrophoresis(PAGE), or on agarose gels.

IV. HIGH RESOLUTION POSITIONING OF CENTROMERES ON THE GENETIC MAP

Southern genomic DNA blots in combination with RFLP analysis may be usedto map centromeres with a high degree of resolution. The stored seedlingtissue provides the necessary amount of DNA for analysis of therestriction fragments. Southern blots are hybridized to probes labeledby radioactive or non-radioactive methods.

It may, in many cases, be desired to identify new polymorphic DNAmarkers which are closely linked to the target region. In some casesthis can be readily done. For example when comparing Landsberg andColumbia DNA, a polymorphic Sau3A site can be found for about every 8 to20 kB surveyed. Subtractive methods are available for identifying suchpolymorphisms (Rosenberg et al., 1994), and these subtractions may beperformed using DNA from selected, centromeric YAC clones. Screens forRFLP markers potentially linked to centromeres also can be performedusing DNA fragments from a centromere-linked YAC clone to probe blots ofLandsberg and Columbia genomic DNA that has been digested with a panelof restriction enzymes.

V. ISOLATION OF CENTROMERE CONTAINING DNA FRAGMENTS

Using the markers flanking each centromere (see FIG. 3) it is possibleto purify a contiguous DNA fragment that contains both flanking markersand the centromere encoded between those markers. In order to carry thisout, very large DNA fragments up to the size of an entire chromosome areprepared by embedding Arabidopsis tissues in agarose using, for example,the method described by Copenhaver et al., (1995). These large pieces ofDNA can be digested in the agarose with any restriction enzyme. Thoserestriction enzymes which will be particularly useful for isolatingintact centromeres include enzymes which yield very large DNA fragments.Such restriction enzymes include those with specificities greater thansix base pairs such as, for example, Asc I, Bae I, BbvC I, Fse I, Not I,Pac I, Pme I, PpuM I, Rsr II, SanD I, Sap I, SexA I, Sfi I, Sgf I, SgrAI, Sbf I, Srf I, Sse8387 I, Sse8647 I, Swa, UbaD I, and UbaE I, or anyother enzyme that cuts at a low frequency within the Arabidopsis genome,and specifically within the centromeric region. Alternatively, a partialdigest with a more frequent cutting restriction enzyme could be used.

The large DNA fragments produced by digestion with restriction enzymesare then separated by size using pulsed-field gel electrophoresis (PFGE)(Schwartz et al., 1982). Specifically, Contour-clamped HomogeneousElectric Field (CHEF) electrophoresis (a variety of PFGE) can be used toseparate DNA molecules as large as 10 Mb (Chu et al., 1985). Large DNAfragments resolved on CHEF gels can then be analyzed using standardSouthern hybridization techniques to identify and measure the size ofthose fragments which contain both centromere flanking markers andtherefor, the centromere. After determining the size of the centromerecontaining fragment by comparison with known size standards, the regionfrom the gel that contains the centromere fragment can be cut out of aduplicate gel. This centromeric DNA can then be analyzed, sequenced, andused in a variety of applications, as described below, including theconstruction of plant artificial chromosomes (PLACs). As indicated indetail below, PLACs can be constructed by attaching telomeres andselectable markers to the centromere fragment cut from the agarose gelusing standard techniques which allow DNA ligation within the gel slice.Plant cells can then be transformed with this hybrid DNA molecule usingthe techniques described herein below.

VI. PLAC CONSTRUCTS

In light of the instant disclosure it will be possible for those ofordinary skill in the art to construct the artificial chromosomesdescribed herein. Useful construction methods are well-known (see, forexample, Maniatis et al., 1982). As constructed, the PLAC willpreferably include at least an autonomous replication sequence (ARS)functional in plants, a centromere functional in plants, and telomereswhich are functional in plants.

In addition to the basic elements, positive and negative selectableplant markers (e.g., antibiotic or herbicide resistance genes), and acloning site for insertion of foreign DNA will preferably be included.In addition, a visible marker, such as green fluorescent protein, alsomay be desirable. In order to propagate the vectors in E. coli, it isnecessary to convert the linear molecule into a circle by addition of astuffer fragment between the telomeres. Inclusion of an E. coli plasmidreplication origin and selectable marker also may be preferred. It alsomay be desirable to include Agrobacterium sequences to improvereplication and transfer to plant cells. Exemplary artificial chromosomeconstructs are given in FIGS. 7A-7H, although it will be apparent tothose in skill art that many changes may be made in the order and typesof elements present in these constructs and still obtain a functionalartificial chromosome within the scope of the instant invention.

Artificial plant chromosomes which replicate in yeast also may beconstructed to take advantage of the large insert capacity and stabilityof repetitive DNA inserts afforded by this system (see Burke et al.,1987). In this case, yeast ARS and CEN sequences are added to thevector. The artificial chromosome is maintained in yeast as a circularmolecule using a stuffer fragment to separate the telomeres.

A fragment of DNA, from any source whatsoever, may be purified andinserted into an artificial plant chromosome at any appropriaterestriction endonuclease cleavage site. The DNA segment usually willinclude various regulatory signals for the expression of proteinsencoded by the fragment. Alternatively, regulatory signals resident inthe artificial chromosome may be utilized.

The techniques and procedures required to accomplish insertion arewell-known in the art (see Maniatis et al., 1982). Typically, this isaccomplished by incubating a circular plasmid or a linear DNA fragmentin the presence of a restriction endonuclease such that the restrictionendonuclease cleaves the DNA molecule. Endonucleases preferentiallybreak the internal phosphodiester bonds of polynucleotide chains. Theymay be relatively unspecific, cutting polynucleotide bonds regardless ofthe surrounding nucleotide sequence. However, the endonucleases whichcleave only a specific nucleotide sequence are called restrictionenzymes. Restriction endonucleases generally internally cleave DNAmolecules at specific recognition sites, making breaks within“recognition” sequences that in many, but not all, cases exhibittwo-fold symmetry around a given point. Such enzymes typically createdouble-stranded breaks.

Many of these enzymes make a staggered cleavage, yielding DNA fragmentswith protruding single-stranded 5′ or 3′ termini. Such ends are said tobe “sticky” or “cohesive” because they will hydrogen bond tocomplementary 3′ or 5′ ends. As a result, the end of any DNA fragmentproduced by an enzyme, such as EcoRI, can anneal with any other fragmentproduced by that enzyme. This properly allows splicing of foreign genesinto plasmids, for example. Some restriction endonucleases that may beparticularly useful with the current invention include HindIII, PstI,EcoRI, and BamHI.

Some endonucleases create fragments that have blunt ends, that is, thatlack any protruding single strands. An alternative way to create bluntends is to use a restriction enzyme that leaves overhangs, but to fillin the overhangs with a polymerase, such as klenow, thereby resulting inblunt ends. When DNA has been cleaved with restriction enzymes that cutacross both strands at the same position, blunt end ligation can be usedto join the fragments directly together. The advantage of this techniqueis that any pair of ends may be joined together, irrespective ofsequence.

Those nucleases that preferentially break off terminal nucleotides arereferred to as exonucleases. For example, small deletions can beproduced in any DNA molecule by treatment with an exonuclease whichstarts from each 3′ end of the DNA and chews away single strands in a 3′to 5′ direction, creating a population of DNA molecules withsingle-stranded fragments at each end, some containing terminalnucleotides. Similarly, exonucleases that digest DNA from the 5′ end orenzymes that remove nucleotides from both strands have often been used.Some exonucleases which may be particularly useful in the presentinvention include Bal31, SI, and ExoIII. These nucleolytic reactions canbe controlled by varying the time of incubation, the temperature, andthe enzyme concentration needed to make deletions. Phosphatases andkinases also may be used to control which fragments have ends which canbe joined. Examples of useful phosphatases include shrimp alkalinephosphatase and calf intestinal alkaline phosphatase. An example of auseful kinase is T4 polynucleotide kinase.

Once the source DNA sequences and vector sequences have been cleaved andmodified to generate appropriate ends they are incubated together withenzymes capable of mediating the ligation of the two DNA molecules.Particularly useful enzymes for this purpose include T4 ligase, E. coliligase, or other similar enzymes. The action of these enzymes results inthe sealing of the linear DNA to produce a larger DNA moleculecontaining the desired fragment (see, for example, U.S. Pat. Nos.4,237,224; 4,264,731; 4,273,875; 4,322,499 and 4,336,336, which arespecifically incorporated herein by reference).

It is to be understood that the termini of the linearized plasmid andthe termini of the DNA fragment being inserted must be complementary orblunt in order for the ligation reaction to be successful. Suitablecomplementarity can be achieved by choosing appropriate restrictionendonucleases (i.e., if the fragment is produced by the same restrictionendonuclease or one that generates the same overhang as that used tolinearize the plasmid, then the termini of both molecules will becomplementary). As discussed previously, in a preferred embodiment, atleast two classes of the vectors used in the present invention areadapted to receive the foreign oligonucleotide fragments in only oneorientation. After joining the DNA segment to the vector, the resultinghybrid DNA can then be selected from among the large population ofclones or libraries.

A method useful for the molecular cloning of DNA sequences includes invitro joining of DNA segments, fragmented from a source high molecularweight genomic DNA, to vector DNA molecules capable of independentreplication. The cloning vector may include plasmid DNA (see Cohen etal, 1973), phage DNA (see Thomas et al, 1974), SV40 DNA (see Nussbaum etal., 1976), yeast DNA, E. coli DNA and most significantly, plant DNA.

A variety of processes are known which may be utilized to effecttransformation; i.e., the inserting of a heterologous DNA sequences intoa host cell, whereby the host becomes capable of efficient expression ofthe inserted sequences.

VII. DEFINITIONS

By “transformation” or “transfection” is meant the acquisition in cellsof new DNA sequences through incorporation of added DNA. This is theprocess by which naked DNA, DNA coated with protein, or whole artificialchromosomes are introduced into a cell, resulting in a heritable change.

By “gene” is meant a DNA sequence that contains information forconstruction of a polypeptide or protein, and includes 5′ and 3′ ends.This also includes genes which encode only RNA products such as tRNA orrRNA genes.

As used herein, “eukaryote” refers to living organisms whose cellscontain nuclei. A eukaryote may be distinguished from a “prokaryote”which is an organism which lacks nuclei. Prokaryotes and eukaryotesdiffer fundamentally in the way their genetic information is organized,as well as their patterns of RNA and protein synthesis.

By the term “lower eukaryote” is meant a eukaryote characterized by acomparatively simple physiology and composition, and most oftenunicellularity. Examples of lower eukaryotes include flagellates,ciliates, and yeast.

By contrast, the term “higher eukaryote” means a multicellulareukaryote, typically characterized by its greater complex physiologicalmechanisms and relatively large size. Generally, complex organisms suchas plants and animals are included in this category. Preferred highereukaryotes to be transformed by the present invention include, forexample, monocot and dicot angiosperm species, gymnosperm species, fernspecies, plant tissue culture cells of these species, animal cells andalgal cells. It will of course be understood that prokaryotes andeukaryotes alike may be transformed by the methods of this invention.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant calli, and the like, as well as whole plantsregenerated therefrom.

As used herein, “heterologous gene” or “foreign gene” is a structuralgene that is foreign, i.e., originating from a donor different from thehost or a chemically synthesized gene, and can include a donor of adifferent species from the host. The heterologous gene codes for apolypeptide or RNA ordinarily not produced by the organism susceptibleto transformation by the expression vehicle. Another type of“heterologous gene” is an altered gene from the host itself, or anunaltered gene which is present in one or more extra copies. One exampleof such an altered gene useful in the present invention is a mutant genewhich encodes a herbicide-resistant form of a normally occurring enzyme.

By “host” is meant any organism that is the recipient of a replicableplasmid, or expression vector comprising an artificial chromosome.Ideally, host strains used for cloning experiments should be free of anyrestriction enzyme activity that might degrade the foreign DNA used.Preferred examples of host cells for cloning, useful in the presentinvention, are bacteria such as Escherichia coli, Bacillus subtilis,Pseudomonas, Streptomyces, Salmonella, and yeast cells such as S.cerevisiae. Host cells which can be targeted for expression of anartificial chromosome may be plant cells of any source and specificallyinclude Arabidopsis, maize, rice, sugarcane, sorghum, barley, soybeans,tobacco, wheat, tomato, potato, citrus, or any other agronomically orscientifically important species.

By “expression” is meant the process by which a structural gene producesan RNA molecule, typically termed messenger RNA (mRNA). The mRNA istypically, but not always, translated into polypeptide(s).

By “linker” it is meant a DNA molecule, generally up to 50 or 60nucleotides long and synthesized chemically, or cloned from othervectors. In a preferred embodiment, this fragment contains one, orpreferably more than one, restriction enzyme site for a blunt-cuttingenzyme and a staggered-cutting enzyme, such as BamHI. One end of thelinker fragment is adapted to be ligatable to one end of the linearmolecule and the other end is adapted to be ligatable to the other endof the linear molecule.

As used herein, a “library” is a pool of random DNA fragments which arecloned. In principle, any gene can be isolated by screening the librarywith a specific hybridization probe (see, for example, Young et al.,1977). Each library may contain the DNA of a given organism inserted asdiscrete restriction enzyme-generated fragments or as randomly sheeredfragments into many thousands of plasmid vectors. For purposes of thepresent invention, E. coli, yeast, and Salmonella plasmids areparticularly useful when the genome inserts come from other organisms.

By “hybridization” is meant the pairing of complementary RNA and DNAstrands to produce an RNA-DNA hybrid, or alternatively, the pairing oftwo DNA single strands from genetically different or the same sources toproduce a double stranded DNA molecule.

The term “plasmid” or “cloning vector” as used herein refers to a closedcovalently circular extrachromosomal DNA or linear DNA which is able toautonomously replicate in a host cell and which is normally nonessentialto the survival of the cell. A wide variety of plasmids and othervectors are known and commonly used in the art (see, for example, Cohenet al., U.S. Pat. No. 4,468,464, which discloses examples of DNAplasmids, and which is specifically incorporated herein by reference).

As used herein, a “probe” is any biochemical reagent (usually tagged insome way for ease of identification), used to identify or isolate agene, a gene product, a DNA segment or a protein.

By “PLAC” it is meant a plant artificial chromosome of the currentinvention, as specifically disclosed herein.

A “selectable marker” is a gene whose presence results in a clearphenotype, and most often a growth advantage for cells that contain themarker. This growth advantage may be present under standard conditions,altered conditions such as elevated temperature, or in the presence ofcertain chemicals such as herbicides or antibiotics. Use of selectablemarkers is described, for example, in Broach et al. (1979). Examples ofselectable markers include the thymidine kinase gene, the cellularadenine-phosphoribosyltransferase gene and the dihydrylfolate reductasegene, hygromycin phosphotransferase genes, the bar gene and neomycinphosphotransferase genes, among others. Preferred selectable markers inthe present invention include genes whose expression confer antibioticor herbicide resistance to the host cell, sufficient to enable themaintenance of a vector within the host cell, and which facilitate themanipulation of the plasmid into new host cells. Of particular interestin the present invention are proteins conferring cellular resistance toampicillin, chloramphenicol, tetracycline, G-418, bialaphos, andglyphosate for example.

By “PLAC-encoded protein” it is meant a polypeptide which is encoded bya sequence of a PLAC of the current invention. This includes thoseproteins encoded by functional sequences of the PLAC, such as selectablemarkers, telomeres, etc., as well as those proteins encoded byheterologous genes of the PLAC.

By “PLAC-associated protein” it is meant a protein encoded by a sequenceof the PLAC or a protein which is encoded by host DNA and binds withrelatively high affinity to the centromeres of the current invention.

VIII. ARABIDOPSIS THALIANA CENTROMERIC DNA SEGMENTS AS HYBRIDIZATIONPROBES AND PRIMERS

In addition to their use in the construction of PLACs, the centromericregions disclosed herein also have a variety of other uses. For example,they also have utility as probes or primers in nucleic acidhybridization embodiments. As such, it is contemplated that nucleic acidsegments that comprise a sequence region that consists of at least a 14nucleotide long contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous DNA segment of acentromere of the current invention will find particular utility. Longercontiguous identical or complementary sequences, e.g., those of about20, 30, 40, 50, 100, 200, 500, 1000, 2000, 5000 bp, etc. including allintermediate lengths and up to and including the full-length sequence ofa centromere of the current invention also will be of use in certainembodiments.

The ability of such nucleic acid probes to specifically hybridize tocentromeric sequences will enable them to be of use in detecting thepresence of similar, partially complementary sequences from other plantsor animals. However, other uses are envisioned, including the use of thecentromeres for the preparation of mutant species primers, or primersfor se in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100 or even of 101-200 nucleotides or so, identical orcomplementary to DNA sequences of a centromere of the current invention,are particularly contemplated as hybridization probes for use in, e.g.,Southern and Northern blotting. Smaller fragments will generally finduse in hybridization embodiments, wherein the length of the contiguouscomplementary region may be varied, such as between about 10-14 andabout 100 or 200 nucleotides, but larger contiguous complementaritystretches also may be used, according to the length complementarysequences one wishes to detect.

Of course, fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. Nos. 4,683,195 and4,683,202 (each incorporated herein by reference), by introducingselected sequences into recombinant vectors for recombinant production,and by other recombinant DNA techniques generally known to those ofskill in the art of molecular biology.

Accordingly, the centromeres of the current invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.15 M NaCl at temperatures ofabout 50° C. to about 70° C. Such selective conditions tolerate little,if any, mismatch between the probe and the template or target strand,and would be particularly suitable for isolating centromeric DNAsegments. Detection of DNA segments via hybridization is well-known tothose of skill in the art, and the teachings of U.S. Pat. Nos. 4,965,188and 5,176,995 (each specifically incorporated herein by reference in itsentirety) are exemplary of the methods of hybridization analyses.Teachings such as those found in the texts of Maloy et al., 1991; Segal1976; Prokop 1991; and Kuby 1994, are particularly relevant.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate centromerefunction-encoding sequences from related species, functionalequivalents, or the like, less stringent hybridization conditions willtypically be needed in order to allow formation of the heteroduplex. Inthese circumstances, one may desire to employ conditions such as about0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. toabout 55° C. Cross-hybridizing species can thereby be readily identifiedas positively hybridizing signals with respect to controlhybridizations. In any case, it is generally appreciated that conditionscan be rendered more stringent by the addition of increasing amounts ofform amide, which serves to destabilize the hybrid duplex in the samemanner as increased temperature or decreased salt. Thus, hybridizationconditions can be readily manipulated, and thus will generally be amethod of choice depending on the desired results.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmentally undesirable reagents. In the case of enzyme tags,colorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

IX. PLAC-ASSOCIATED PROTEIN SPECIFIC ANTIBODY COMPOSITIONS AND METHODSOF MAKING

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which bind toPLAC-associated proteins of the current invention. Such PLAC-associatedproteins include proteins which are coded by the sequences of the PLAC,as well as proteins encoded by host DNA that bind to the centromeres ofthe current invention. It is specifically contemplated that thesePLAC-associated protein specific antibodies would allow for the furtherisolation and characterization of the PLAC-associated proteins. Forexample, proteins may be isolated which are encoded by the centromeres.Recombinant production of such proteins provides a source of antigen forproduction of antibodies.

Alternatively, the centromere may be used as a ligand to isolate, usingaffinity methods, centromere binding proteins. Once isolated, theseprotein can be used as antigens for the production polyclonal andmonoclonal antibodies. A variation on this technique has beendemonstrated by Rattner (1991), by cloning of centromere-associatedproteins through the use of antibodies which bind in the vicinity of thecentromere.

Means for preparing and characterizing antibodies are well known in theart (see, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference). The methods forgenerating monoclonal antibodies (mAbs) generally begin along the samelines as those for preparing polyclonal antibodies. Briefly, apolyclonal antibody is prepared by immunizing an animal with animmunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of antisera is a rabbit, a mouse, a rat, a hamster,a guinea pig or a goat. A rabbit is a preferred choice for production ofpolyclonal antibodies because of the ease of handling, maintenance andrelatively large blood volume.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin also canbe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection also may be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

Monoclonal antibodies may be readily prepared through use of well-knowntechniques, such as those exemplified in U.S. Pat. No. 4,196,265,incorporated herein by reference. Typically, this technique involvesimmunizing a suitable animal with a selected immunogen composition,e.g., a purified or partially purified PLAC-associated protein,polypeptide or peptide. The immunizing composition is administered in amanner effective to stimulate antibody producing cells. Rodents such asmice and rats are preferred animals, however, the use of rabbit, sheep,or frog cells also is possible. The use of rats may provide certainadvantages (Goding 1986), but mice are preferred, with the BALB/c mousebeing most preferred as this is most routinely used and generally givesa higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding 1986; Campbell 1984). For example, where theimmunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653,NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all usefulin connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler et al.,1975; 1976), and those using polyethylene glycol (PEG), such as 37%(v/v) PEG, (Gefter et al., 1977). The use of electrically induced fusionmethods also is appropriate (Goding 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines also could be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

X. ELISAS AND IMMUNOPRECIPITATION

ELISAs may be used in conjunction with the invention. Particularly, itis contemplated that ELISAs will find use in assays of PLAC geneexpression. In an ELISA assay, proteins or peptides comprisingPLAC-encoded protein antigen sequences are immobilized onto a selectedsurface, preferably a surface exhibiting a protein affinity such as thewells of a polystyrene microtiter plate. After washing to removeincompletely adsorbed material, it is desirable to bind or coat theassay plate wells with a nonspecific protein that is known to beantigenically neutral with regard to the test antisera such as bovineserum albumin (BSA), casein or solutions of milk powder. This allows forblocking of nonspecific adsorption sites on the immobilizing surface andthus reduces the background caused by nonspecific binding of antiseraonto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween®. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from about 2 to about 4 hours, at temperatures preferably on theorder of about 25 to about 27° C. Following incubation, theantisera-contacted surface is washed so as to remove non-immunocomplexedmaterial. A preferred washing procedure includes washing with a solutionsuch as PBS/Tween®, or borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound antigen, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for the first. To provide adetecting means, the second antibody will preferably have an associatedenzyme that will generate color or light development upon incubatingwith an appropriate chromogenic substrate. Thus, for example, one willdesire to contact and incubate the antisera-bound surface with a ureaseor peroxidase-conjugated anti-human IgG for a period of time and underconditions which favor the development of immunocomplex formation (e.g.,incubation for 2 hours at room temperature in a PBS-containingsolution).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectra spectrophotometer.

XI. WESTERN BLOTS

The compositions of the present invention may find use in immunoblot orwestern blot analysis. The antibodies of the invention may be used ashigh-affinity primary reagents for the identification of proteinsimmobilized onto a solid support matrix, such as nitrocellulose, nylonor combinations thereof. In conjunction with immunoprecipitation,followed by gel electrophoresis, these may be used as a single stepreagent for use in detecting antigens against which secondary reagentsused in the detection of the antigen cause an adverse background. Thisis especially useful when the antigens studied are immunoglobulins(precluding the use of immunoglobulins binding bacterial cell wallcomponents), the antigens studied cross-react with the detecting agent,or they migrate at the same relative molecular weight as across-reacting signal.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the protein moiety areconsidered to be of particular use in this regard.

XII. DNA SEGMENTS

Further aspects of the present invention concern isolated DNA segmentsand recombinant vectors encoding a functional Arabidopsis thalianacentromere and other sequences for the creation and use of recombinantsequences of a PLAC.

The present invention concerns DNA segments, isolatable from A. thalianacells, that are enriched relative to total genomic DNA and are capableof conferring centromere activity to a recombinant molecule whenincorporated into the host cell. As used herein, the term centromereactivity indicates the ability to confer stable inheritance upon a DNAsegment, artificial chromosome or chromosome.

As used herein, the term “DNA segment” refers to a DNA molecule that hasbeen purified from total genomic DNA of a particular species. Therefore,a DNA segment encoding centromere function refers to a DNA segment thatcontains centromere coding sequences yet is isolated away from, orpurified free from, total genomic DNA of A. thaliana. Included withinthe term “DNA segment”, are DNA segments and smaller fragments of suchsegments, and also recombinant vectors, including, for example,plasmids, cosmids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified centromericsequence refers to a DNA segment including centromere coding sequencesand, in certain aspects, regulatory sequences, isolated substantiallyaway from other naturally occurring genes, protein encoding sequences,or other DNA sequences. In this respect, the term “gene” is used forsimplicity to refer to a functional DNA segment, protein, polypeptide orpeptide encoding unit. As will be understood by those in the art, thisfunctional term includes both genomic sequences, cDNA sequences andsmaller engineered gene segments that may express, or may be adapted toexpress, proteins, polypeptides or peptides.

“Isolated substantially away from other coding sequences” means that thesequences of interest, in this case centromere function encodingsequences, are included within the genomic DNA clones provided herein.Of course, this refers to the DNA segment as originally isolated, anddoes not exclude genes or coding regions later added to the segment bythe hand of man.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences that encode acentromere functional sequence that includes a contiguous sequence fromthe centromeres of the current invention. In certain other embodiments,the invention concerns isolated DNA segments and recombinant vectorsthat include within their sequence a contiguous nucleic acid sequencefrom an A. thaliana centromere. Again, DNA segments that exhibitcentromere function activity will be most preferred.

The nucleic acid segments of the present invention, regardless of thelength of the sequence itself, may be combined with other DNA sequences,such as promoters, polyadenylation signals, additional restrictionenzyme sites, multiple cloning sites, other coding segments, and thelike, such that their overall length may vary considerably. It istherefore contemplated that a nucleic acid fragment of almost any lengthmay be employed, with the total length preferably being limited by theease of preparation and use in the intended recombinant DNA protocol.

XIII. BIOLOGICAL FUNCTIONAL EQUIVALENTS

Modification and changes may be made in the centromeric DNA segments ofthe current invention and still obtain a functional molecule withdesirable characteristics. The following is a discussion based uponchanging the nucleic acids of a centromere to create an equivalent, oreven an improved, second-generation molecule.

In particular embodiments of the invention, mutated centromericsequences are contemplated to be useful for increasing the utility ofthe centromere. It is specifically contemplated that the function of thecentromeres of the current invention may be based upon the secondarystructure of the DNA sequences of the centromere and/or the proteinswhich interact with the centromere. By changing the DNA sequence of thecentromere, one may alter the affinity of one or morecentromere-associated protein(s) for the centromere and/or the secondarystructure of the centromeric sequences, thereby changing the activity ofthe centromere. Alternatively, changes may be made in the centromeres ofthe invention which do not effect the activity of the centromere.Changes in the centromeric sequences which reduce the size of the DNAsegment needed to confer centromere activity are contemplated to beparticularly useful in the current invention, as would changes whichincreased the fidelity with which the centromere was transmitted duringmitosis and meiosis.

XIV. OBTAINING A. THALIANA CENTROMERIC DNA FROM YEAST AND BACTERIALCLONES

The Arabidopsis physical map consists primarily of YAC clones between200 kb and 1 Mb in length, and an overlapping contig map with severalanchors to the genetic map is available (Hwang et al., 1991)(http://cbil.humgen.upenn.edu/atgc/ATGCUP.html). To be certain that anentire centromeric region has been cloned, clones or a series of clones,are identified that hybridize to markers on either side of eachcentromere. These efforts can be complicated by the presence ofrepetitive DNA in the centromere, as well as by the potentialinstability of centromere clones. Thus, identification of large YACswith unique sequences that will serve as useful probes simplifies achromosome walking strategy. Use of BACs may also be advantageous, asdata has suggested that YAC clones may sometimes not span centromeres(Willard, 1997).

Blot hybridization allows comparison of the structure of the clones withthat of genomic DNA, and thus determines whether the clones havesuffered deletions or rearrangements. The centromeric clones identifiedare useful for hybridization experiments that can be used to determinewhether they share common sequences, whether they localize in situ tothe cytologically defined centromeric region, and whether they containrepetitive sequences thought to map near Arabidopsis centromeres(Richards et al., 1991; Maluszynska et al., 1991).

In a positional cloning approach, one may wish to begin sequencing ifthe region of interest has been narrowed to a sufficiently small area.The determination of what constitutes a sufficiently small sequence isdependent upon many factors including, but not necessarily limited to,the repetitive DNA content of the region of interest, the size of theregion to be sequenced, the ability to obtain higher resolution geneticmapping data, and the relative efficiency of sequencing technology. Inmany cases, sequencing may be begun when the target region has beennarrowed to 40 kB or less. It is estimated that in Arabidopsis thaliana1 cM corresponds to approximately 200 kB (Hwang, 1991; Koornneef, 1987).Such a resolution will on average, be provided by analysis of about 100tetrads for each centromere, although some centromeres may be mappedwith higher resolution due to the fact that each tetrad issimultaneously mapping five centromeres, and thus has five times theusual probability of finding a crossover very close to a locus ofinterest. Nonetheless, because the amount of DNA per cM can increase inthe vicinity of the centromere (Carpenter et al., 1982), examination ofadditional tetrad progeny and identification of additional geneticmarkers may be required to achieve the desired degree of geneticlinkage.

XV. TRANSFORMED HOST CELLS AND TRANSGENIC PLANTS

Methods and compositions for transforming a bacterium, a yeast cell, aplant cell, or an entire plant with one or more artificial chromosomesare further aspects of this disclosure. A transgenic bacterium, yeastcell, plant cell or plant derived from such a transformation process orthe progeny and seeds from such a transgenic plant also are furtherembodiments of the invention.

Means for transforming bacteria and yeast cells are well known in theart Typically, means of transformation are similar to those well knownmeans used to transform other bacteria or yeast such as E. coli orSaccharomyces cerevisiae. Methods for DNA transformation of plant cellsinclude Agrobacterium-mediated plant transformation, protoplasttransformation, gene transfer into pollen, injection into reproductiveorgans, injection into immature embryos and particle bombardment. Eachof these methods has distinct advantages and disadvantages. Thus, oneparticular method of introducing genes into a particular plant strainmay not necessarily be the most effective for another plant strain, butit is well known in the art which methods are useful for a particularplant strain.

There are many methods for introducing transforming DNA segments intocells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as by Agrobacterium infection,direct delivery of DNA such as, for example, by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

Technology for introduction of DNA into cells is well-known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham et al., 1973;Zatloukal et al., 1992); (2) physical methods such as microinjection(Capecchi, 1980), electroporation (Wong et al., 1982; Fromm et al.,1985; U.S. Pat. No. 5,384,253) and the gene gun (Johnston et al., 1994;Fynan et al., 1993); (3) viral vectors (Clapp 1993; Lu et al., 1993;Eglitis et al., 1988a; 1988b); and (4) receptor-mediated mechanisms(Curiel et al., 1991; 1992; Wagner et al., 1992).

(i) Electroporation

The application of brief, high-voltage electric pulses to a variety ofanimal and plant cells leads to the formation of nanometer-sized poresin the plasma membrane. DNA is taken directly into the cell cytoplasmeither through these pores or as a consequence of the redistribution ofmembrane components that accompanies closure of the pores.Electroporation can be extremely efficient and can be used both fortransient expression of cloned genes and for establishment of cell linesthat carry integrated copies of the gene of interest. Electroporation,in contrast to calcium phosphate-mediated transfection and protoplastfusion, frequently gives rise to cell lines that carry one, or at most afew, integrated copies of the foreign DNA.

The introduction of DNA by means of electroporation, is well-known tothose of skill in the art. In this method, certain cell wall-degradingenzymes, such as pectin-degrading enzymes, are employed to render thetarget recipient cells more susceptible to transformation byelectroporation than untreated cells. Alternatively, recipient cells aremade more susceptible to transformation, by mechanical wounding. Toeffect transformation by electroporation one may employ either friabletissues such as a suspension culture of cells, or embryogenic callus, oralternatively, one may transform immature embryos or other organizedtissues directly. One would partially degrade the cell walls of thechosen cells by exposing them to pectin-degrading enzymes (pectolyases)or mechanically wounding in a controlled manner. Such cells would thenbe recipient to DNA transfer by electroporation, which may be carriedout at this stage, and transformed cells then identified by a suitableselection or screening protocol dependent on the nature of the newlyincorporated DNA.

(ii) Microprojectile Bombardment

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming monocots, is thatneither the isolation of protoplasts (Cristou et al., 1988) nor thesusceptibility to Agrobacterium infection is required. An illustrativeembodiment of a method for delivering DNA into maize cells byacceleration is a Biolistics Particle Delivery System, which can be usedto propel particles coated with DNA or cells through a screen, such as astainless steel or Nytex screen, onto a filter surface covered withplant cells cultured in suspension. The screen disperses the particlesso that they are not delivered to the recipient cells inlarge-aggregates. It is believed that a screen intervening between theprojectile apparatus and the cells to be bombarded reduces the size ofprojectiles aggregate and may contribute to a higher frequency oftransformation by reducing damage inflicted on the recipient cells byprojectiles that are too large.

For the bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens also arepositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth herein one may obtainup to 1000 or more foci of cells transiently expressing a marker gene.The number of cells in a focus which express the exogenous gene product48 hours post-bombardment often range from 1 to 10 and average 1 to 3.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance, and helium pressure.One also may minimize the trauma reduction factors (TRFs) by modifyingconditions which influence the physiological state of the recipientcells and which may therefore influence transformation and integrationefficiencies. For example, the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient cells may be adjustedfor optimum transformation. The execution of other routine adjustmentswill be known to those of skill in the art in light of the presentdisclosure.

(iii) Agrobacterium-Mediated Transfer

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al., 1987). Using conventional transformationvectors, chromosomal integration is required for stable inheritance ofthe foreign DNA. However, the vector described herein may be used fortransformation with or without integration, as the centromere functionrequired for stable inheritance is encoded within the PLAC. Inparticular embodiments, transformation events in which the PLAC is notchromosomally integrated may be preferred, in that problems withsite-specific variations in expression and insertional mutagenesis maybe avoided.

The integration of the Ti-DNA is a relatively precise process resultingin few rearrangements. The region of DNA to be transferred is defined bythe border sequences, and intervening DNA is usually inserted into theplant genome as described (Spielmann et al., 1986; Jorgensen et al.,1987). Modern Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al, 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described (Rogers et al,1987), have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

Agrobacterium-mediated transformation of leaf disks and other tissuessuch as cotyledons and hypocotyls appears to be limited to plants thatAgrobacterium naturally infects. Agrobacterium-mediated transformationis most efficient in dicotyledonous plants. Few monocots appear to benatural hosts for Agrobacterium, although transgenic plants have beenproduced in asparagus and more significantly in maize usingAgrobacterium vectors as described (Bytebier et al., 1987; U.S. Pat. No.5,591,616, specifically incorporated herein by reference). Therefore,commercially important cereal grains such as rice, corn, and wheat mustusually be transformed using alternative methods. However, as mentionedabove, the transformation of asparagus using Agrobacterium also can beachieved (see, for example, Bytebier et al., 1987).

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome. Such transgenicplants can be referred to as being hemizygous for the added gene. A moreaccurate name for such a plant is an independent segregant, because eachtransformed plant represents a unique T-DNA integration event.

More preferred is a transgenic plant that is homozygous for the addedforeign DNA; i.e., a transgenic plant that contains two copies of atransgene, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by sexually mating(selfing) an independent segregant transgenic plant that contains asingle added transgene, germinating some of the seed produced andanalyzing the resulting plants produced for enhanced activity relativeto a control (native, non-transgenic) or an independent segreganttransgenic plant.

Even more preferred is a plant in which the PLAC has not beenchromosomally integrated. Such a plant may be termed 2n+x, where 2n isthe diploid number of chromosomes and where x is the number of PLACs.Initially, transformants may be 2n+1, i.e. having 1 additional PLAC. Inthis case, it may be desirable to self the plant or to cross the plantwith another 2n+1 plant to yield a plant which is 2n+2. The 2n+2 plantis preferred in that it is expected to pass the PLAC through meiosis toall its offspring.

It is to be understood that two different transgenic plants also can bemated to produce offspring that contain two independently segregatingadded, exogenous PLACs. Selfing of appropriate progeny can produceplants that are homozygous for both added, exogenous PLACs that encode apolypeptide of interest. Back-crossing to a parental plant andout-crossing with a non-transgenic plant also are contemplated.

XVI. OTHER TRANSFORMATION METHODS

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1986; Uchimiyaet al, 1986; Callis et al., 1987; Marcotte et al., 1988).

Application of these systems to different plant strains for the purposeof making transgenic plants depends upon the ability to regenerate thatparticular plant strain from protoplasts. Illustrative methods for theregeneration of cereals from protoplasts are described (Fujimura et al.,1985; Toriyama et al., 1986; Yamada et al., 1986; Abdullah et al.,1986).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil 1988). Inaddition, “particle gun” or high-velocity microprojectile technology canbe utilized (Vasil 1992).

Using that latter technology, DNA is carried through the cell wall andinto the cytoplasm on the surface of small metal particles as described(Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988). The metalparticles penetrate through several layers of cells and thus allow thetransformation of cells within tissue explants.

XVII. EXOGENOUS GENES FOR EXPRESSION IN PLANTS

One particularly important advance of the present invention is that itprovides methods and compositions for expression of exogenous genes inplant cells. Significantly, the current invention allows for thetransformation of plant cells with a PLAC comprising a number ofexogenous genes. Such genes often will be genes that direct theexpression of a particular protein or polypeptide product, but they alsomay be non-expressible DNA segments, e.g., transposons such as Ds thatdo not direct their own transposition. As used herein, an “expressiblegene” is any gene that is capable of being transcribed into RNA (e.g.,mRNA, antisense RNA, etc.) or translated into a protein, expressed as atrait of interest, or the like, etc., and is not limited to selectable,screenable or non-selectable marker genes. The inventors alsocontemplate that, where both an expressible gene that is not necessarilya marker gene is employed in combination with a marker gene, one mayemploy the separate genes on either the same or different DNA segmentsfor transformation. In the latter case, the different vectors aredelivered concurrently to recipient cells to maximize cotransformation.

The choice of the particular DNA segments to be delivered to therecipient cells often will depend on the purpose of the transformation.One of the major purposes of transformation of crop plants is to addsome commercially desirable, agronomically important traits to theplant. Such traits include, but are not limited to, herbicide resistanceor tolerance; insect resistance or tolerance; disease resistance ortolerance (viral, bacterial, fungal, nematode); stress tolerance and/orresistance, as exemplified by resistance or tolerance to drought, heat,chilling, freezing, excessive moisture, salt stress; oxidative stress;increased yields; food content and makeup; physical appearance; malesterility; drydown; standability; prolificacy; starch quantity andquality; oil quantity and quality; protein quality and quantity; aminoacid composition; and the like. One may desire to incorporate one ormore genes conferring any such desirable trait or traits, such as, forexample, a gene or genes encoding herbicide resistance.

In certain embodiments, the present invention contemplates thetransformation of a recipient cell with PLACs comprising more than oneexogenous gene. As used herein, an “exogenous gene,” is a gene notnormally found in the host genome in an identical context. By this, itis meant that the gene may be isolated from a different species thanthat of the host genome, or alternatively, isolated from the host genomebut operably linked to one or more regulatory regions which differ fromthose found in the unaltered, native gene. Two or more exogenous genesalso can be supplied in a single transformation event using eitherdistinct transgene-encoding vectors, or using a single vectorincorporating two or more gene coding sequences. For example, plasmidsbearing the bar and aroA expression units in either convergent,divergent, or colinear orientation, are considered to be particularlyuseful. Further preferred combinations are those of an insect resistancegene, such as a Bt gene, along with a protease inhibitor gene such aspinII, or the use of bar in combination with either of the above genes.Of course, any two or more transgenes of any description, such as thoseconferring herbicide, insect, disease (viral, bacterial, fungal,nematode) or drought resistance, male sterility, drydown, standability,prolificacy, starch properties, oil quantity and quality, or thoseincreasing yield or nutritional quality may be employed as desired.

(i) Herbicide Resistance

The genes encoding phosphinothricin acetyltransferase (bar and pat),glyphosate tolerant EPSP synthase genes, the glyphosate degradativeenzyme gene gox encoding glyphosate oxidoreductase, deh (encoding adehalogenase enzyme that inactivates dalapon), herbicide resistant(e.g., sulfonylurea and imidazolinone) acetolactate synthase, and bxngenes (encoding a nitrilase enzyme that degrades bromoxynil) are goodexamples of herbicide resistant genes for use in transformation. The barand pat genes code for an enzyme, phosphinothricin acetyltransferase(PAT), which inactivates the herbicide phosphinothricin and preventsthis compound from inhibiting glutamine synthetase enzymes. The enzyme5-enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase), is normallyinhibited by the herbicide N-(phosphonomethyl)glycine (glyphosate).However, genes are known that encode glyphosate-resistant EPSP synthaseenzymes. These genes are particularly contemplated for use in planttransformation. The deh gene encodes the enzyme dalapon dehalogenase andconfers resistance to the herbicide dalapon. The bxn gene codes for aspecific nitrilase enzyme that converts bromoxynil to a non-herbicidaldegradation product.

(ii) Insect Resistance

Potential insect resistance genes that can be introduced includeBacillus thuringiensis crystal toxin genes or Bt genes (Watrud et al.,1985). Bt genes may provide resistance to lepidopteran or coleopteranpests such as European Corn Borer (ECB). Preferred Bt toxin genes foruse in such embodiments include the CryIA(b) and CryIA(c) genes.Endotoxin genes from other species of B. thuringiensis which affectinsect growth or development also may be employed in this regard.

It is contemplated that preferred Bt genes for use in the transformationprotocols disclosed herein will be those in which the coding sequencehas been modified to effect increased expression in plants, and moreparticularly, in monocot plants. Means for preparing synthetic genes arewell known in the art and are disclosed in, for example, U.S. Pat. No.5,500,365 and U.S. Pat. No. 5,689,052, each of the disclosures of whichare specifically incorporated herein by reference in their entirety.Examples of such modified Bt toxin genes include a synthetic Bt CryIA(b)gene (Perlak et al., 1991), and the synthetic CryIA(c) gene termed 1800b(PCT Application WO 95/06128). Some examples of other Bt toxin genesknown to those of skill in the art are given in Table 1 below.

TABLE 1 Bacillus thuringiensis δ-Endotoxin Genes^(a) New NomenclatureOld Nomenclature GenBank Accession Cry1Aa CryIA(a) M11250 Cry1AbCryIA(b) M13898 Cry1Ac CryIA(c) M11068 Cry1Ad CryIA(d) M73250 Cry1AeCryIA(e) M65252 Cry1Ba CryIB X06711 Cry1Bb ET5 L32020 Cry1Bc PEG5 Z46442Cry1Bd CryE1 U70726 Cry1Ca CryIC X07518 Cry1Cb CryIC(b) M97880 Cry1DaCryID X54160 Cry1Db PrtB Z22511 Cry1Ea CryIE X53985 Cry1Eb CryIE(b)M73253 Cry1Fa CryIF M63897 Cry1Fb PrtD Z22512 Cry1Ga PrtA Z22510 Cry1GbCryH2 U70725 Cry1Ha PrtC Z22513 Cry1Hb U35780 Cry1Ia CryV X62821 Cry1IbCryV U07642 Cry1Ja ET4 L32019 Cry1Jb ET1 U31527 Cry1K U28801 Cry2AaCryIIA M31738 Cry2Ab CryIIB M23724 Cry2Ac CryIIC X57252 Cry3A CryIIIAM22472 Cry3Ba CryIIIB X17123 Cry3Bb CryIIIB2 M89794 Cry3C CryIIID X59797Cry4A CryIVA Y00423 Cry4B CryIVB X07423 Cry5Aa CryVA(a) L07025 Cry5AbCryVA(b) L07026 Cry6A CryVIA L07022 Cry6B CryVIB L07024 Cry7Aa CryIIICM64478 Cry7Ab CryIIICb U04367 Cry8A CryIIIE U04364 Cry8B CryIIIG U04365Cry8C CryIIIF U04366 Cry9A CryIG X58120 Cry9B CryIX X75019 Cry9C CryIHZ37527 Cry10A CryIVC M12662 Cry11A CryIVD M31737 Cry11B Jeg80 X86902Cry12A CryVB L07027 Cry13A CryVC L07023 Cry14A CryVD U13955 Cry15A 34kDa M76442 Cry16A cbm71 X94146 Cry17A cbm71 X99478 Cry18A CryBP1 X99049Cry19A Jeg65 Y08920 Cyt1Aa CytA X03182 Cyt1Ab CytM X98793 Cyt2A CytBZ14147 Cyt2B CytB U52043 ^(a)Adapted from:http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html

Protease inhibitors also may provide insect resistance (Johnson et al.,1989), and will thus have utility in plant transformation. The use of aprotease inhibitor II gene, pinII, from tomato or potato is envisionedto be particularly useful. Even more advantageous is the use of a pinIIgene in combination with a Bt toxin gene, the combined effect of whichhas been discovered to produce synergistic insecticidal activity. Othergenes which encode inhibitors of the insect's digestive system, or thosethat encode enzymes or co-factors that facilitate the production ofinhibitors, also may be useful. This group may be exemplified byoryzacystatin and amylase inhibitors such as those from wheat andbarley.

Also, genes encoding lectins may confer additional or alternativeinsecticide properties. Lectins (originally termed phytohemagglutinins)are multivalent carbohydrate-binding proteins which have the ability toagglutinate red blood cells from a range of species. Lectins have beenidentified recently as insecticidal agents with activity againstweevils, ECB and rootworm (Murdock et al., 1990; Czapla & Lang, 1990).Lectin genes contemplated to be useful include, for example, barley andwheat germ agglutinin (WGA) and rice lectins (Gatehouse et al., 1984),with WGA being preferred.

Genes controlling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as, e.g.,lytic peptides, peptide hormones and toxins and venoms, form anotheraspect of the invention. For example, it is contemplated that theexpression of juvenile hormone esterase, directed towards specificinsect pests, also may result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock et al., 1990).

Transgenic plants expressing genes which encode enzymes that affect theintegrity of the insect cuticle form yet another aspect of theinvention. Such genes include those encoding, e.g., chitinase,proteases, lipases and also genes for the production of nikkomycin, acompound that inhibits chitin synthesis, the introduction of any ofwhich is contemplated to produce insect resistant plants. Genes thatcode for activities that affect insect molting, such as those affectingthe production of ecdysteroid UDP-glucosyl transferase, also fall withinthe scope of the useful transgenes of the present invention.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host plant to insect pestsalso are encompassed by the present invention. It may be possible, forinstance, to confer insecticidal activity on a plant by altering itssterol composition. Sterols are obtained by insects from their diet andare used for hormone synthesis and membrane stability. Thereforealterations in plant sterol composition by expression of novel genes,e.g., those that directly promote the production of undesirable sterolsor those that convert desirable sterols into undesirable forms, couldhave a negative effect on insect growth and/or development and henceendow the plant with insecticidal activity. Lipoxygenases are naturallyoccurring plant enzymes that have been shown to exhibit anti-nutritionaleffects on insects and to reduce the nutritional quality of their diet.Therefore, further embodiments of the invention concern transgenicplants with enhanced lipoxygenase activity which may be resistant toinsect feeding.

Tripsacum dactyloides is a species of grass that is resistant to certaininsects, including corn root worm. It is anticipated that genes encodingproteins that are toxic to insects or are involved in the biosynthesisof compounds toxic to insects will be isolated from Tripsacum and thatthese novel genes will be useful in conferring resistance to insects. Itis known that the basis of insect resistance in Tripsacum is genetic,because said resistance has been transferred to Zea mays via sexualcrosses (Branson and Guss, 1972). It is further anticipated that othercereal, monocot or dicot plant species may have genes encoding proteinsthat are toxic to insects which would be useful for producing insectresistant plants.

Further genes encoding proteins characterized as having potentialinsecticidal activity also may be used as transgenes in accordanceherewith. Such genes include, for example, the cowpea trypsin inhibitor(CPTI; Hilder et al., 1987) which may be used as a rootworm deterrent;genes encoding avermectin (Avermectin and Abamectin, Campbell, W. C.,Ed., 1989; Ikeda et al., 1987) which may prove particularly useful as acorn rootworm deterrent; ribosome inactivating protein genes; and evengenes that regulate plant structures. Transgenic plants includinganti-insect antibody genes and genes that code for enzymes that canconvert a non-toxic insecticide (pro-insecticide) applied to the outsideof the plant into an insecticide inside the plant also are contemplated.

(iii) Environment or Stress Resistance

Improvement of a plants ability to tolerate various environmentalstresses such as, but not limited to, drought, excess moisture,chilling, freezing, high temperature, salt, and oxidative stress, alsocan be effected through expression of novel genes. It is proposed thatbenefits may be realized in terms of increased resistance to freezingtemperatures through the introduction of an “antifreeze” protein such asthat of the Winter Flounder (Cutler et al., 1989) or synthetic genederivatives thereof. Improved chilling tolerance also may be conferredthrough increased expression of glycerol-3-phosphate acetyltransferasein chloroplasts (Wolter et al., 1992). Resistance to oxidative stress(often exacerbated by conditions such as chilling temperatures incombination with high light intensities) can be conferred by expressionof superoxide dismutase (Gupta et al., 1993), and may be improved byglutathione reductase (Bowler et al., 1992). Such strategies may allowfor tolerance to freezing in newly emerged fields as well as extendinglater maturity higher yielding varieties to earlier relative maturityzones.

It is contemplated that the expression of novel genes that favorablyeffect plant water content, total water potential, osmotic potential,and turgor will enhance the ability of the plant to tolerate drought. Asused herein, the terms “drought resistance” and “drought tolerance” areused to refer to a plants increased resistance or tolerance to stressinduced by a reduction in water availability, as compared to normalcircumstances, and the ability of the plant to function and survive inlower-water environments. In this aspect of the invention it isproposed, for example, that the expression of genes encoding for thebiosynthesis of osmotically-active solutes, such as polyol compounds,may impart protection against drought. Within this class are genesencoding for mannitol-L-phosphate dehydrogenase (Lee and Saier, 1982)and trehalose-6-phosphate synthase (Kaasen et al., 1992). Through thesubsequent action of native phosphatases in the cell or by theintroduction and coexpression of a specific phosphatase, theseintroduced genes will result in the accumulation of either mannitol ortrehalose, respectively, both of which have been well documented asprotective compounds able to mitigate the effects of stress. Mannitolaccumulation in transgenic tobacco has been verified and preliminaryresults indicate that plants expressing high levels of this metaboliteare able to tolerate an applied osmotic stress (Tarczynski et al, 1992,1993).

Similarly, the efficacy of other metabolites in protecting either enzymefunction (e.g., alanopine or propionic acid) or membrane integrity(e.g., alanopine) has been documented (Loomis et al., 1989), andtherefore expression of genes encoding for the biosynthesis of thesecompounds might confer drought resistance in a manner similar to orcomplimentary to mannitol. Other examples of naturally occurringmetabolites that are osmotically active and/or provide some directprotective effect during drought and/or desiccation include fructose,erythritol (Coxson et al., 1992), sorbitol, dulcitol (Karsten et al.,1992), glucosylglycerol (Reed et al., 1984; ErdMann et al, 1992),sucrose, stachyose (Koster and Leopold, 1988; Blackman et al, 1992),raffinose (Bernal-Lugo and Leopold, 1992), proline (Rensburg et al.,1993), glycine betaine, ononitol and pinitol (Vernon and Bohnert, 1992).Continued canopy growth and increased reproductive fitness during timesof stress will be augmented by introduction and expression of genes suchas those controlling the osmotically active compounds discussed aboveand other such compounds. Currently preferred genes which promote thesynthesis of an osmotically active polyol compound are genes whichencode the enzymes mannitol-1-phosphate dehydrogenase,trehalose-6-phosphate synthase and myoinositol 0-methyltransferase.

It is contemplated that the expression of specific proteins also mayincrease drought tolerance. Three classes of Late Embryogenic Proteinshave been assigned based on structural similarities (see Dure et al,1989). All three classes of LEAs have been demonstrated in maturing(i.e. desiccating) seeds. Within these 3 types of LEA proteins, theType-II (dehydrin-type) have generally been implicated in drought and/ordesiccation tolerance in vegetative plant parts (i.e. Mundy and Chua,1988; Piatkowski et al., 1990; Yamaguchi-Shinozaki et al, 1992).Recently, expression of a Type-III LEA (HVA-1) in tobacco was found toinfluence plant height, maturity and drought tolerance (Fitzpatrick,1993). In rice, expression of the HVA-1 gene influenced tolerance towater deficit and salinity (Xu et al., 1996). Expression of structuralgenes from all three LEA groups may therefore confer drought tolerance.Other types of proteins induced during water stress include thiolproteases, aldolases and transmembrane transporters (Guerrero et al.,1990), which may confer various protective and/or repair-type functionsduring drought stress. It also is contemplated that genes that effectlipid biosynthesis and hence membrane composition might also be usefulin conferring drought resistance on the plant.

Many of these genes for improving drought resistance have complementarymodes of action. Thus, it is envisaged that combinations of these genesmight have additive and/or synergistic effects in improving droughtresistance in plants. Many of these genes also improve freezingtolerance (or resistance); the physical stresses incurred duringfreezing and drought are similar in nature and may be mitigated insimilar fashion. Benefit may be conferred via constitutive expression ofthese genes, but the preferred means of expressing these novel genes maybe through the use of a turgor-induced promoter (such as the promotersfor the turgor-induced genes described in Guerrero et al, 1990 andShagan et al, 1993 which are incorporated herein by reference). Spatialand temporal expression patterns of these genes may enable plants tobetter withstand stress.

It is proposed that expression of genes that are involved with specificmorphological traits that allow for increased water extractions fromdrying soil would be of benefit. For example, introduction andexpression of genes that alter root characteristics may enhance wateruptake. It also is contemplated that expression of genes that enhancereproductive fitness during times of stress would be of significantvalue. For example, expression of genes that improve the synchrony ofpollen shed and receptiveness of the female flower parts, i.e., silks,would be of benefit. In addition it is proposed that expression of genesthat minimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value.

Given the overall role of water in determining yield, it is contemplatedthat enabling plants to utilize water more efficiently, through theintroduction and expression of novel genes, will improve overallperformance even when soil water availability is not limiting. Byintroducing genes that improve the ability of plants to maximize waterusage across a full range of stresses relating to water availability,yield stability or consistency of yield performance may be realized.

(iv) Disease Resistance

It is proposed that increased resistance to diseases may be realizedthrough introduction of genes into plants, for example, intomonocotyledonous plants such as maize. It is possible to produceresistance to diseases caused by viruses, bacteria, fungi and nematodes.It also is contemplated that control of mycotoxin producing organismsmay be realized through expression of introduced genes.

Resistance to viruses may be produced through expression of novel genes.For example, it has been demonstrated that expression of a viral coatprotein in a transgenic plant can impart resistance to infection of theplant by that virus and perhaps other closely related viruses (Cuozzo etal., 1988, Hemenway et al., 1988, Abel et al, 1986). It is contemplatedthat expression of antisense genes targeted at essential viral functionsmay also impart resistance to viruses. For example, an antisense genetargeted at the gene responsible for replication of viral nucleic acidmay inhibit replication and lead to resistance to the virus. It isbelieved that interference with other viral functions through the use ofantisense genes also may increase resistance to viruses. Further, it isproposed that it may be possible to achieve resistance to virusesthrough other approaches, including, but not limited to the use ofsatellite viruses.

It is proposed that increased resistance to diseases caused by bacteriaand fungi may be realized through introduction of novel genes. It iscontemplated that genes encoding so-called “peptide antibiotics,”pathogenesis related (PR) proteins, toxin resistance, and proteinsaffecting host-pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences which are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in monocotyledonousplants such as maize may be useful in conferring resistance to bacterialdisease. These genes are induced following pathogen attack on a hostplant and have been divided into at least five classes of proteins (Bol,Linthorst, and Comelissen, 1990). Included amongst the PR proteins areβ-1,3-glucanases, chitinases, and osmotin and other proteins that arebelieved to function in plant resistance to disease organisms. Othergenes have been identified that have antifungal properties, e.g., UDA(stinging nettle lectin) and hevein (Broakaert et al., 1989;Barkai-Golan et al., 1978). It is known that certain plant diseases arecaused by the production of phytotoxins. It is proposed that resistanceto these diseases would be achieved through expression of a novel genethat encodes an enzyme capable of degrading or otherwise inactivatingthe phytotoxin. It also is contemplated that expression of novel genesthat alter the interactions between the host plant and pathogen may beuseful in reducing the ability of the disease organism to invade thetissues of the host plant, e.g., an increase in the waxiness of the leafcuticle or other morphological characteristics.

(v) Plant Agronomic Characteristics

Two of the factors determining where crop plants can be grown are theaverage daily temperature during the growing season and the length oftime between frosts. Within the areas where it is possible to grow aparticular crop, there are varying limitations on the maximal time it isallowed to grow to maturity and be harvested. For example, a variety tobe grown in a particular area is selected for its ability to mature anddry down to harvestable moisture content within the required period oftime with maximum possible yield. Therefore, crops of varying maturitiesis developed for different growing locations. Apart from the need to drydown sufficiently to permit harvest, it is desirable to have maximaldrying take place in the field to minimize the amount of energy requiredfor additional drying post-harvest. Also, the more readily a productsuch as grain can dry down, the more time there is available for growthand kernel fill. It is considered that genes that influence maturityand/or dry down can be identified and introduced into plant lines usingtransformation techniques to create new varieties adapted to differentgrowing locations or the same growing location, but having improvedyield to moisture ratio at harvest. Expression of genes that areinvolved in regulation of plant development may be especially useful.

It is contemplated that genes may be introduced into plants that wouldimprove standability and other plant growth characteristics. Expressionof novel genes in plants which confer stronger stalks, improved rootsystems, or prevent or reduce ear droppage would be of great value tothe farmer. It is proposed that introduction and expression of genesthat increase the total amount of photoassimilate available by, forexample, increasing light distribution and/or interception would beadvantageous. In addition, the expression of genes that increase theefficiency of photosynthesis and/or the leaf canopy would furtherincrease gains in productivity. It is contemplated that expression of aphytochrome gene in crop plants may be advantageous. Expression of sucha gene may reduce apical dominance, confer semidwarfism on a plant, andincrease shade tolerance (U.S. Pat. No. 5,268,526). Such approacheswould allow for increased plant populations in the field.

(vi) Nutrient Utilization

The ability to utilize available nutrients may be a limiting factor ingrowth of crop plants. It is proposed that it would be possible to alternutrient uptake, tolerate pH extremes, mobilization through the plant,storage pools, and availability for metabolic activities by theintroduction of novel genes. These modifications would allow a plantsuch as maize to more efficiently utilize available nutrients. It iscontemplated that an increase in the activity of, for example, an enzymethat is normally present in the plant and involved in nutrientutilization would increase the availability of a nutrient. An example ofsuch an enzyme would be phytase. It is further contemplated thatenhanced nitrogen utilization by a plant is desirable. Expression of aglutamate dehydrogenase gene in plants, e.g., E. coli gdhA genes, maylead to increased fixation of nitrogen in organic compounds.Furthermore, expression of gdhA in plants may lead to enhancedresistance to the herbicide glufosinate by incorporation of excessammonia into glutamate, thereby detoxifying the ammonia. It also iscontemplated that expression of a novel gene may make a nutrient sourceavailable that was previously not accessible, e.g., an enzyme thatreleases a component of nutrient value from a more complex molecule,perhaps a macromolecule.

(vii) Male Sterility

Male sterility is useful in the production of hybrid seed. It isproposed that male sterility may be produced through expression of novelgenes. For example, it has been shown that expression of genes thatencode proteins that interfere with development of the maleinflorescence and/or gametophyte result in male sterility. Chimericribonuclease genes that express in the anthers of transgenic tobacco andoilseed rape have been demonstrated to lead to male sterility (Marianiet al., 1990).

A number of mutations were discovered in maize that confer cytoplasmicmale sterility. One mutation in particular, referred to as T cytoplasm,also correlates with sensitivity to Southern corn leaf blight A DNAsequence, designated TURF-13 (Levings, 1990), was identified thatcorrelates with T cytoplasm. It is proposed that it would be possiblethrough the introduction of TURF-13 via transformation, to separate malesterility from disease sensitivity. As it is necessary to be able torestore male fertility for breeding purposes and for grain production,it is proposed that genes encoding restoration of male fertility alsomay be introduced.

(viii) Negative Selectable Markers

Introduction of genes encoding traits that can be selected against maybe useful for eliminating PLACs from a cell or for selecting againstcells which comprise a particular PLAC. An example of a negativeselectable marker which has been investigated is the enzyme cytosinedeaminase (Stouggard, 1993). In the presence of this enzyme the compound5-fluorocytosine is converted to 5-fluorouracil which is toxic to plantand animal cells. Therefore, cells comprising a PLAC with this genecould be directly selected against. Other genes that encode proteinsthat render the plant sensitive to a certain compound will also beuseful in this context. For example, T-DNA gene 2 from Agrobacteriumtumefaciens encodes a protein that catalyzes the conversion ofα-naphthalene acetamide (NAM) to α-naphthalene acetic acid (NAA) rendersplant cells sensitive to high concentrations of NAM (Depicker et al.,1988).

(ix) Non-Protein-Expressing Sequences

DNA may be introduced into plants for the purpose of expressing RNAtranscripts that function to affect plant phenotype yet are nottranslated into protein. Two examples are antisense RNA and RNA withribozyme activity. Both may serve possible functions in reducing oreliminating expression of native or introduced plant genes. However, asdetailed below, DNA need not be expressed to effect the phenotype of aplant.

1. Antisense RNA

Genes may be constructed or isolated, which when transcribed, produceantisense RNA that is complementary to all or part(s) of a targetedmessenger RNA(s). The antisense RNA reduces production of thepolypeptide product of the messenger RNA. The polypeptide product may beany protein encoded by the plant genome. The aforementioned genes willbe referred to as antisense genes. An antisense gene may thus beintroduced into a plant by transformation methods to produce a noveltransgenic plant with reduced expression of a selected protein ofinterest. For example, the protein may be an enzyme that catalyzes areaction in the plant. Reduction of the enzyme activity may reduce oreliminate products of the reaction which include any enzymaticallysynthesized compound in the plant such as fatty acids, amino acids,carbohydrates, nucleic acids and the like. Alternatively, the proteinmay be a storage protein, such as a zein, or a structural protein, thedecreased expression of which may lead to changes in seed amino acidcomposition or plant morphological changes respectively. Thepossibilities cited above are provided only by way of example and do notrepresent the full range of applications.

2. Ribozymes

Genes also may be constructed or isolated, which when transcribed,produce RNA enzymes (ribozymes) which can act as endoribonucleases andcatalyze the cleavage of RNA molecules with selected sequences. Thecleavage of selected messenger RNAs can result in the reduced productionof their encoded polypeptide products. These genes may be used toprepare novel transgenic plants which possess them. The transgenicplants may possess reduced levels of polypeptides including, but notlimited to, the polypeptides cited above.

Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987;Forster and Symons, 1987). For example, a large number of ribozymesaccelerate phosphoester transfer reactions with a high degree ofspecificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes.

Several different ribozyme motifs have been described with RNA cleavageactivity (Symons, 1992). Examples include sequences from the Group Iself splicing introns including Tobacco Ringspot Virus (Prody et al.,1986), Avocado Sunblotch Viroid (Palukaitis et al., 1979; Symons, 1981),and Lucerne Transient Streak Virus (Forster and Symons, 1987). Sequencesfrom these and related viruses are referred to as hammerhead ribozymebased on a predicted folded secondary structure.

Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan et al., 1992, Yuan and Altman, 1994, U.S. Pat.Nos. 5,168,053 and 5,624,824), hairpin ribozyme structures(Berzal-Herranz et al., 1992; Chowrira et al., 1993) and Hepatitis Deltavirus based ribozymes (U.S. Pat. No. 5,625,047). The general design andoptimization of ribozyme directed RNA cleavage activity has beendiscussed in detail (Haseloff and Gerlach, 1988, Symons, 1992, Chowriraet al., 1994; Thompson et al., 1995).

The other variable on ribozyme design is the selection of a cleavagesite on a given target RNA. Ribozymes are targeted to a given sequenceby virtue of annealing to a site by complimentary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence which is the cleavage site. For hammerhead ribozyme,the cleavage site is a dinucleotide sequence on the target RNA is auracil (U) followed by either an adenine, cytosine or uracil (A, C or U)(Perriman et al., 1992; Thompson et al., 1995). The frequency of thisdinucleotide occurring in any given RNA is statistically 3 out of 16.Therefore, for a given target messenger RNA of 1000 bases, 187dinucleotide cleavage sites are statistically possible.

Designing and testing ribozymes for efficient cleavage of a target RNAis a process well known to those skilled in the art. Examples ofscientific methods for designing and testing ribozymes are described byChowrira et al., (1994) and Lieber and Strauss (1995), each incorporatedby reference. The identification of operative and preferred sequencesfor use in down regulating a given gene is simply a matter of preparingand testing a given sequence, and is a routinely practiced “screening”method known to those of skill in the art.

3. Induction of Gene Silencing

It also is possible that genes may be introduced to produce noveltransgenic plants which have reduced expression of a native gene productby the mechanism of co-suppression. It has been demonstrated in tobacco,tomato, and petunia (Goring et al., 1991; Smith et al, 1990; Napoli etal, 1990; van der Krol et al, 1990) that expression of the sensetranscript of a native gene will reduce or eliminate expression of thenative gene in a manner similar to that observed for antisense genes.The introduced gene may encode all or part of the targeted nativeprotein but its translation may not be required for reduction of levelsof that native protein.

4. Non-RNA-Expressing Sequences

DNA elements including those of transposable elements such as Ds, Ac, orMu, may be inserted into a gene to cause mutations. These DNA elementsmay be inserted in order to inactivate (or activate) a gene and thereby“tag” a particular trait. In this instance the transposable element doesnot cause instability of the tagged mutation, because the utility of theelement does not depend on its ability to move in the genome. Once adesired trait is tagged, the introduced DNA sequence may be used toclone the corresponding gene, e.g., using the introduced DNA sequence asa PCR primer together with PCR gene cloning techniques (Shapiro, 1983;Dellaporta et al., 1988). Once identified, the entire gene(s) for theparticular trait, including control or regulatory regions where desired,may be isolated, cloned and manipulated as desired. The utility of DNAelements introduced into an organism for purposes of gene tagging isindependent of the DNA sequence and does not depend on any biologicalactivity of the DNA sequence, i.e., transcription into RNA ortranslation into protein. The sole function of the DNA element is todisrupt the DNA sequence of a gene.

It is contemplated that unexpressed DNA sequences, including novelsynthetic sequences, could be introduced into cells as proprietary“labels” of those cells and plants and seeds thereof. It would not benecessary for a label DNA element to disrupt the function of a geneendogenous to the host organism, as the sole function of this DNA wouldbe to identify the origin of the organism. For example, one couldintroduce a unique DNA sequence into a plant and this DNA element wouldidentify all cells, plants, and progeny of these cells as having arisenfrom that labeled source. It is proposed that inclusion of label DNAswould enable one to distinguish proprietary germplasm or germplasmderived from such, from unlabelled germplasm.

Another possible element which may be introduced is a matrix attachmentregion element (MAR), such as the chicken lysozyme A element (Stief,1989), which can be positioned around an expressible gene of interest toeffect an increase in overall expression of the gene and diminishposition dependent effects upon incorporation into the plant genome(Stief et al., 1989; Phi-Van et al., 1990).

XVIII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skilled the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Generation of an Arabidopsis thaliana Mapping Population

To generate a pollen donor plant, two alleles of qrtl were crossed toone another. The qrtl-1 allele was in the Landsberg ecotype backgroundand the qrtl-2 allele was in the Columbia ecotype background. TheLandsberg ecotype was readily discernible from the Columbia ecotypebecause it carries a recessive mutation, erecta, which causes the stemsto thicken, infloresences to be more compact, and the leaves to be morerounded and small than wildtype. To utilize this as a marker of a donorplant, qrtl-2 pollen was crossed onto a qrtl-1 female stigma. The F₁progeny were heterozygous at all molecular markers yet the progenyretain the quartet phenotype of a tetrad of fused pollen grains. Inaddition, progeny display the ERECTA phenotype of the Columbia plant.This visible marker serves as an indication that the crossing wassuccessful in generating plants segregating ecotype specific markers.Further testing was done to the donor plants by performing PCR analysisto insure that progeny were heterozygous at molecular loci.

Due to the fact that the pollen grains can not be directly assayed formarker segregation and because of the desire to create a long-termresource available for multiple marker assays, it was necessary to crossindividual tetrads generated by the donor plant. This created sets ofprogeny plants which yielded both large quantities of tissue and seed.These crosses were accomplished efficiently by generating a recipientplant homozygous for glaborous 1-1 and male sterility. The recessivevisible mutant, glaborous1-1, was chosen to guard against thepossibility of the recipient plant self-fertilizing and the progenybeing mistaken for tetrad plants. Pollen inviability through malesterile-1 was introduced to guard against the recipient plantself-fertilizing. Due to the fact that the homozygous plant does notself, a stock seed generated by a heterozygous male sterility 1 plantneeds to be maintained from which sterile recipient plants can beselected. A large resource of seed segregating the optimal sterile andvisually marked recipient plant was obtained.

Example 2 Tetrad Pollinations

Tetrad pollinations were carried out as follows. A mature flower wasremoved from the donor plant and tapped upon a glass microscope slide torelease mature tetrad pollen grains. This slide was then placed under a20-40× Zeiss dissecting microscope. To isolate individual tetrad pollengrains, a small wooden dowel was used to which an eyebrow hair withrubber cement was mounted. Using the light microscope, a tetrad pollenunit was chosen and touched to the eyebrow hair. The tetradpreferentially adhered to the eyebrow hair and was thus lifted from themicroscope slide and transported the recipient plant stigmatic surface.The transfer was carried out without the use of the microscope, and theeyebrow hair with adhering tetrad was then placed against the recipientstigmatic surface and the hair was manually dragged across the stigmasurface. The tetrad then preferentially adhered to the stigma of therecipient and the cross pollination was completed.

Preferably, an additional backcross of the qrtl-2 parent was used toincrease the pollination efficiency, which was increased to >80%successful seed set production. Initially, 57 tetrad seed setsconsisting of 34 seeds each, were collected. Plants were grown fromthese tetrad seed sets, and tissue was collected. DNA was extracted froma small portion of the stored tissue for PCR based segregation analysis.Additionally the segregation of the visible erecta phenotype was scored.When the plants set seed, the seed was collected as a source for thelarger amounts of DNA required to analyze RFLP segregation by Southernblotting.

Example 3 Genetic Mapping of Centromeres

To map centromeres, F₁ plants which were heterozygous for hundreds ofpolymorphic DNA markers were generated by crossing quartet mutants fromthe Landsberg and Columbia ecotypes (Chang et al. 1988; Ecker, 1994;Konieczy and Ausubel, 1993). In tetrads from these plants, geneticmarkers segregate in a 2:2 ratio (FIG. 6; Preuss et al. 1994). Thesegregation of markers was then determined in plants which weregenerated by crossing pollen grains from the F₁ plants onto a Landsberghomozygote. The genotype of the pollen grains within a tetrad wasinferred from the genotype of the progeny. Initially, seeds weregenerated from greater than 100 successful tetrad pollinations, andtissue and seeds were collected from 57 of these. This providedsufficient material for PCR, as well as seeds necessary for producingthe large quantities of tissue required for Southern hybridization andRFLP mapping. In order to obtain a more precise localization of thecentromeres the original tetrad population was increased from 57 tetradsto over 388 tetrads. Additional tetrads may be collected to provide evenbetter resolution.

PCR analysis was performed to determine marker segregation. To accountfor the contribution of the Landsberg background from the female parent,one Landsberg complement from each of the four tetrad plants wassubtracted. As shown in FIG. 5, markers from sites spanning the entiregenome were used for pair-wise comparisons of all other markers.Tetratypes indicate a crossover between one or both markers and theircentromeres where as ditypes indicate the absence of crossovers (orpresence of a double crossover).

Thus, at every genetic locus, the resulting diploid progeny was eitherL/C or C/C. The map generated with these plants is based solely on malemeioses, unlike the existing map, which represents an average ofrecombination's in both males and females. Therefore, severalwell-established genetic distances were recalculated and thus willdetermine whether recombination frequencies are significantly altered.

The large quantities of genetic data generated by the analysis must becompared pair-wise to perform tetrad analysis. All of the data wasmanaged in a Microsoft Excel spread-sheet format, assigning Landsbergalleles a value of “1” and Columbia alleles a value of “0”. Within atetrad, the segregation of markers on one chromosome was compared tocentromere-linked reference loci on a different chromosome (see Table 2below). Multiplying the values of each locus by an appropriatereference, and adding the results for each tetrad easily distinguishedPD, NPD, and TT tetrads with values of 2, 0, and 1, respectively. Byscoring more than 53 PCR-based genetic markers distributed across thegenome, all five Arabidopsis thaliana centromeres were mapped to smallintervals (FIG. 3A-3E). Additionally, for each centromeric interval, anumber of useful recombinants were identified.

TABLE 2 Scoring protocol for tetratypes. Individual Refer- members enceReference Reference of a tetrad Locus 1 Locus Locus 2 Locus Locus 3Locus A 1 × 1 = 1 0 × 1 = 0 0 × 1 = 0 B 1 × 1 = 1 0 × 1 = 0 1 × 1 = 1 C0 × 0 = 0 1 × 0 = 0 0 × 0 = 0 D 0 × 0 = 0 1 × 0 = 0 1 × 0 = 0 — 2 0 1 PDNPD TT

Example 4 Mapping Results Arabidopsis Chromosomes 1-5

The centromere on chromosome 1 was mapped to between UFO (47.5 cM) andGAPB (59 cM). A more refined position places the centromere between themarker 7G6 (˜55 cM) and T27K12 (˜59 cM) however, since neither of thesemarkers is precisely mapped, the centromere may be between the followingpublicly available marker pairs: UFO and ACBP, ACBP AND Ve009, Ve009 andm254A, m254A and p39B2T7, p39B2T7 and m253, m253 and Ve0101, Ve0101 andmi423a, m423a and RPS18B, RPS18B and AIG1, AIG1 and mi63, mi63 and mi19,mi19 and agP6e, agP6e and mi342, mi342 and EKRIV, EKRIV and intel1-1,intel1-1 and EKRIII, EKRIII and mi133, or mi133 and GAPB.

The centromere on chromosome 1 was further mapped between mi342 (56.7cM) and T27K12 (59.1 cM). A more refined position places the centromerebetween the marker T22C23 (−58.5 cM) and T27K12 (59.1 cM). However,since T22C23 is not precisely mapped the centromere may be between thepublicly available markers mi342 and T27K12.

The centromere on chromosome 2 was mapped between m246 (11.1 cM) andm216 (33.3). A more refined position places the centromere between themarkers m246 (11.1 cM) and THY1B (˜30 cM) however, since THY1B is notprecisely mapped, the centromere may lie between the following publiclyavailable marker pairs: m246 and m497A, m497A and g4553, g4553 and RNS1,RNS1 and Ve013, Ve013 and Cds3, Cds3 and mi310, mi310 and EKRII-C,EKRII-C and EKRII, EKRII and mi444, mi444 and mi421, mi421 and g4532,g4532 and SEP2A and g4133, g4133 and PR1, PR1 and mi398, or mi398 andm216.

The centromere on chromosome 2 was further mapped between mi310 (18.6cM) and g4133 (23.8 cM). Within this interval, the centromere may bebetween the following publicly available marker pairs: mi310 and mi421,mi421 and g4532, g4532 and SEP2A, and SEP2A and g4133.

The centromere on chromosome 3 was mapped to between GL1 (44.7 cM) andTOPP5 (55.6 cM). A more refined position places the centromere betweenGL1 (44.7cM) and NIT1 (−55 cM) however, since NIT1 is not preciselymapped, the centromere may be between the following publicly availablemarker pairs: GL1 and BRC1, BRC1 and AIG2, AIG2 and mi413, mi413 andatpox, atpox and mi358, mi358 and mi79b, mi79b and EKRI-B, EKRI-B andASD, ASD and a-1, a-1 and t04109 and ve012, or ve012 and TOPP5.

The centromere on chromosome 3 was further mapped between atpox (48.6cM) and ve021 (54.7 cM). A more refined position places the centromerebetween the marker atpox (48.6 cM) and 91F1T7 (˜54.2 cM). However, since91F1T7 is not precisely mapped, the centromere may be between thefollowing publicly available marker pairs: atpox and zim2, zim2 andmi79b, mi79b and RCEN3, RCEN3 and ASD, ASD and a-1, a-1 and t04109, andt04109 and ve021.

The centromere on chromosome 4 was mapped between GA1 (16.7 cM) and nga8(24.3 cM). Within this interval the centromere may be between thepublicly available marker pairs GA1 and petc, petc and Cds13, Cds13 andm4848A, m448A and mi233, mi233A and g2616, g2616 and m506, m506 andmi306, mi306 and nga12, nga12 and BIO200, BIO200 and Cl lath, Cl lathand m456A, and m456A and mi87, mi87 and mi167, mi167 and EKRI-A, orEKR1-A and nga8.

The centromere on chromosome 4 was further mapped between mi233 (18.8cM) and mi167 (21.5 cM). A more refined position places the centromerebetween the markers mi233 and F13H14. However, since F13H14 is notprecisely mapped, the centromere may be between the following publiclyavailable marker pairs: mi233 and g2616, g2616 and mi306, mi306 andm506, m506 and nga12, nga12 and BIO200, BIO200 and mi87, mi87 andC11Ath, C11Ath and m456A, and m456A and mi167.

The centromere on chromosome 5 was mapped between nga76 (71.6 cM) andPhC (74.3 cM). Within this interval the centromere may be between thefollowing publicly available marker pairs: nga76 and mi291b, mi291b andCMs1, and CMs1 and PhyC.

All of the above markers and genetic positions (i.e. cM values)correspond to the Lister and Dean Recombinant Inbred Genetic map,available on-line at: http://genome-www3.stanford.edu/atdb_welcome.html.

Example 5 Construction of Artificial Plant Chromosomes

Plant artificial chromosomes are constructed by combining the previouslyisolated essential chromosomal elements. Exemplary artificialchromosomes include those designed to be “shuttle vectors”; i.e., theycan be maintained in a convenient host (such as E. coli, Agrobacteriumor yeast) as well as plant cells.

An artificial chromosome can be maintained in E. coli or other bacterialcells as a circular molecule by placing a removable stuffer fragmentbetween the telomeric sequence blocks. The stuffer fragment is adispensable DNA sequence, bordered by unique restriction sites, whichcan be removed by restriction digestion of the circular DNAs to createlinear molecules with telomeric ends. The linear PLAC can then beisolated by, for example, gel electrophoresis. In addition to thestuffer fragment and the plant telomeres, the artificial chromosomecontains a replication origin and selectable marker that can function inplants to allow the circular molecules to be maintained in bacterialcells. The artificial chromosomes also include a plant selectablemarker, a plant centromere, and a plant ARS to allow replication andmaintenance of the DNA molecules in plant cells. Finally, the artificialchromosome includes several unique restriction sites where additionalDNA sequence inserts can be cloned. The most expeditious method ofphysically constructing such an artificial chromosome, i.e., ligatingthe various essential elements together for example, will be apparent tothose of ordinary skill in this art.

A number of artificial chromosome vectors have been designed by thecurrent inventors and are disclosed herein for the purpose ofillustration (FIGS. 7A-7H). These vectors are not limiting however, asit will be apparent to those of skill in the art that many changes andalterations may be made and still obtain a functional vector.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A recombinant DNA construct comprising a Arabidopsis thalianacentromere sequence and at least three structural genes, wherein thecentromere sequence maps to a location on an Arabidopsis thalianachromosome defined by a pair of genetic markers selected from the groupconsisting of UFO and GAPB, m246 and m216, GL1 and TOPP5, GA1 and nga8,and nga76 and PhyC, and wherein the centromere sequence sorts DNA tospindle poles in meiosis 1 in a pattern indicating disjunction ofhomologous chromosomes.
 2. The recombinant DNA construct of claim 1,which additionally comprises a telomere functional in plants.
 3. Therecombinant DNA construct of claim 1, which additionally comprises aselectable marker gene.
 4. The recombinant DNA construct of claim 3,which is capable of being maintained as a chromosome, wherein saidchromosome is stably transmitted in dividing cells.
 5. The recombinantDNA construct of claim 1, which comprises at least five structuralgenes.
 6. The construct of claim 1, wherein at least one of saidstructural genes is selected from the group consisting of an antibioticresistance gene; a herbicide resistance gene, a nitrogen fixation gene;a plant pathogen defense gene; a plant stress-induced gene; a toxingene; and a seed storage gene.
 7. The construct of claim 1, wherein atleast one of said structural genes is selected from the group consistingof a hormone gene; an enzyme gene; an interleukin gene; a clottingfactor gene; a cytokine gene; an antibody gene; and a growth factorgene.
 8. A host cell transformed with the construct of claim
 5. 9. Amethod of expressing a structural gene in a plant comprisingtransforming a regenerable plant cell with the recombinant DNA constructof claim
 1. 10. The method of claim 9, further comprising expressing asecond structural gene.
 11. A plant produced by the method of claim ofclaim 9.